Mutant protease biosensors with enhanced detection characteristics

ABSTRACT

A polynucleotide encoding a biosensor polypeptide comprising a modified circularly-permuted thermostable luciferase and a linker linking the C-terminal portion of the thermostable luciferase to the N-terminal portion of the thermostable luciferase. The modified circularly-permuted thermostable luciferase is modified relative to a parental circularly-permuted thermostable luciferase. The linker contains a sensor region capable of interacting with a target molecule in a cell. The modified circularly-permuted thermostable luciferase has an enhanced response after interaction of the biosensor with the target molecule relative to the parental circularly-permuted thermostable luciferase in the presence of the target molecule. Alternatively, the modified circularly-permuted thermostable luciferase has an enhanced response after interaction of the biosensor with the target molecule relative to the modified circularly-permuted thermostable luciferase in the absence of the target molecule.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/333,706, filed May 11, 2010, and U.S. Provisional PatentApplication Ser. No. 61/470,845, filed Apr. 1, 2011, each of which isincorporated herein by reference in its entirety.

SEQUENCE LISTING

The sequence listing is filed with the application in electronic formatonly and is incorporated herein by reference. The sequence listing textfile “ASFILED_Sequence_US02.txt” was created on May 11, 2011, and is152,798 bytes in size.

FIELD OF INVENTION

The present invention relates to the field of biochemical assays andreagents. More specifically, this invention relates to modifiedluciferases and methods for their use.

BACKGROUND

Luciferases are enzymes that catalyze the oxidation of a substrate(e.g., luciferin or coelenterazine) with the concomitant release ofphotons of light. Luciferases have been isolated from numerous species,including Coleopteran arthropods and many sea creatures as well asbacteria. Because it is easily detectable and its activity can bequantified with high precision, luciferases have been widely used tostudy gene expression and protein localization. Unlike green fluorescentprotein (GFP), which requires up to 30 minutes to form its chromophore,the products of luciferases can be detected immediately upon completionof synthesis of the polypeptide chain (if substrate and oxygen are alsopresent). In addition, no post-translational modifications are requiredfor enzymatic activity, and the enzyme contains no prosthetic groups,bound cofactors, or disulfide bonds. Luciferases are useful reporters innumerous species and in a wide variety of cells.

Luciferases possess additional features that render them particularlyuseful as reporter molecules for biosensing, i.e., molecules whichreveal molecular properties of a system. Most catalytic reactionsgenerate less than the energy of hydrolysis for two molecules of ATP, orabout 70 kJ/mole. However, the luminescence elicited by luciferases hasmuch higher energy content. For instance, the reaction catalyzed byfirefly luciferase (560 nm) emits 214 kJ/mole of energy. Furthermore,luciferases are also highly efficient at converting chemical energy intophotons, i.e., they have high quantum yields. Luciferases are thusextremely efficient for generating detectable signals.

SUMMARY

In one embodiment, the invention provides a polynucleotide encoding abiosensor polypeptide comprising a modified circularly-permutedthermostable luciferase and a linker. The linker links the C-terminalportion of the thermostable luciferase to the N-terminal portion of thethermostable luciferase. The modified circularly-permuted thermostableluciferase is modified relative to a parental circularly-permutedthermostable luciferase. The linker comprises a sensor region capable ofinteracting with a target molecule in a cell. The modifiedcircularly-permuted thermostable luciferase has an enhanced responseafter interaction of the biosensor with the target molecule relative tothe parental circularly-permuted thermostable luciferase in the presenceof the target molecule. Alternatively, the modified circularly-permutedthermostable luciferase has an enhanced response after interaction ofthe biosensor with the target molecule relative to the modifiedcircularly-permuted thermostable luciferase in the absence of the targetmolecule.

In another embodiment, the invention provides a polynucleotide encodinga biosensor polypeptide comprising a modified circularly-permutedthermostable luciferase and a linker, wherein the modifiedcircularly-permuted thermostable luciferase has a substitution of atleast one amino acid at positions 5, 17, 21, 23, 26, 39, 44, 51, 81,101, 103, 110, 114, 115, 119, 123, 126, 128, 133, 137, 186, 191, 192,193, 196, 208, 211, 214, 226, 228, 230, 233, 264, 273, 275, 286, 287,294, 295, 297, 302, 303, 304, 306, 308, 309, 313, 324, 329, 331, 343,348, 353, 364, 374, 385, 389, 409, 420, 426, 427, 428, 431, 449, 456,460, 461, 465, 466, 468, 471, 473, 482, 484, 485, 489, 493, 494, 497,503, 507, 509, 510, 513, 516, 517, 521, 522, 523, 526, 530, 533, 536,537, 542, or 543 corresponding to SEQ ID NO: 2. The linker links theC-terminal portion of the thermostable luciferase to the N-terminalportion of the thermostable luciferase. The linker has a sensor regioncapable of interacting with a target molecule in a cell. The modifiedcircularly-permuted thermostable luciferase has an enhanced responseafter interaction of the biosensor with the target molecule relative tothe parental circularly-permuted thermostable luciferase in the presenceof the target molecule. Alternatively, the modified circularly-permutedthermostable luciferase has an enhanced response after interaction ofthe biosensor with the target molecule relative to the modifiedcircularly-permuted thermostable luciferase in the absence of the targetmolecule. The modified circularly-permuted thermostable luciferase mayalso have increased luminescence or increased stability relative to anunmodified circularly-permuted thermostable luciferase.

In an aspect, the disclosure relates to a method to detect the presenceor activity of a target molecule in a sample, comprising contacting thesample with a polynucleotide encoding a modified circularly-permutedthermostable luciferase biosensor comprising a sensor region for thetarget molecule and a substrate for the modified circularly-permutedthermostable luciferase and detecting luminescence in the sample.

In an aspect, the disclosure relates to a method to detect the presenceor activity of a target molecule in a cell, comprising contacting a cellwith a polynucleotide encoding a modified circularly-permutedthermostable luciferase biosensor comprising a sensor region for thetarget molecule and a substrate for the modified circularly-permutedthermostable luciferase and detecting luminescence in the cell.

In an aspect, the disclosure relates to a method to detect the presenceor activity of a target molecule in an animal, comprising contacting ananimal with a modified circularly-permuted thermostable luciferasebiosensor comprising a sensor region for the target molecule and asubstrate for the modified circularly-permuted thermostable luciferaseand detecting luminescence in the animal.

In an aspect, the disclosure relates to a method to detect the presenceor activity of a target molecule in a sample, comprising immobilizing amodified circularly-permuted thermostable luciferase biosensorcomprising a sensor region for the target molecule to a solid support,adding a sample containing the target molecule to the immobilizedbiosensor, adding a substrate for the modified circularly-permutedthermostable luciferase, and detecting luminescence.

In an aspect, the disclosure relates to a method to detect apoptosis ina sample, comprising contacting the sample with a polynucleotideencoding a modified circularly-permuted thermostable luciferasebiosensor comprising a sensor region for a molecule involved inapoptosis and a substrate for the modified circularly-permutedthermostable luciferase and detecting luminescence in the sample.

Other aspects of the invention will become apparent by consideration ofthe detailed description and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the position of the amino acid substitutions, 1471T,5503G, T5071, and S193P in the corresponding starting sequenceTL-CP358-DEVD:DD.

FIGS. 2A-B show the normalized RLU for the variant 01:A-05 and thecorresponding starting sequence TL-CP358-DEVD:DD after treatment withTNF-α-related apoptosis inducing ligand (TRAIL) treatment (FIG. 2A) andthe fold-induction (response) after 2 and 10 hrs (FIG. 2B).

FIGS. 3A-B show the normalized RLU for the variants FC7:24, FC7:43, andFC7:49, compared to corresponding starting sequence TL-CP358-DEVD:DDafter treatment with TRAIL (FIG. 3A) and the fold-induction (response)after 2 and 10 hrs (FIG. 3B).

FIG. 4 shows the effect of the linker on the performance of theCaspase-3/7 BioSensor (CBS).

FIG. 5 shows the kinetic profile of Caspase 8 activation by TRAIL overtime at 37° C. using TL-CP233-Caspase 8 and TL-CP358-Caspase 8Biosensors.

FIG. 6 shows the fold response of Caspase 8 activation by TRAIL overtime using TL-CP233-Caspase 8 and TL-CP358-Caspase 8 Biosensors.

FIGS. 7A-D show the kinetic profile of Caspase 8 activation by TRAILover time at 37° C. using FF-CP359 Caspase 8 (FIG. 7A), TL-CP233-Caspase8 (FIG. 7B), TL-CP358-Caspase 8 (FIG. 7C), and TL-CP358-Caspase3 (FIG.7C) Biosensors.

FIG. 8 shows fold response of Caspase 8 activation by TRAIL over timeusing TL-CP358-Caspase 3, TL-CP233-Caspase 8, TL-CP358-Caspase 8 andFF-CP359-Caspase 8 Biosensors.

FIG. 9 shows TEV protease Biosensors detect TEV co-expressed in CHOcells.

FIGS. 10A-D show the luminescence (photon counts/sec) of D54-MG cellsexpressing the various thermostable biosensors upon treatment with TRAILat various time points (FIG. 10A), the fold induction (FIG. 10B), theaverage photon counts/sec at baseline, 2, 4 and 6 hrs post treatment(FIG. 10C) and a Western blot showing reporter expression (FIG. 10D).

FIGS. 11A-C show the luminescence (photon counts/sec) of D54-MG reporterxenografted nude mice treated with 8 mg/kg of TRAIL (FIG. 11A), the foldinduction (FIG. 11B), and average photon counts/sec at baseline and 6hrs post treatment (FIG. 11C).

FIGS. 12A-D show the normalized data compared to pre-treatment valuesfor intratibial implanted MDA-MB23101833 cells stably expressingTL-CP233-Caspase 3 treated with TRAIL (FIG. 12A), the Z factorcalculated for every time point (FIG. 12B), representative images takenat the indicated time points (FIG. 12C), and fold induction ofxenografted animals tested treated with TRAIL (FIG. 12D).

FIGS. 13A-D show the relative luminescence upon compound treatment (max)from compounds in the NIH Clinical Collection Biofocus Library (FIG.13A) and the TimTec Kinase Inhibitor Library (FIG. 13C) and the heat mapof data acquired for the NIH Clinical Collection Biofocus Library (FIG.13B) and the TimTec Kinase Inhibitor Library (FIG. 13D).

FIG. 14 shows SDS-PAGE gel analysis of the proteins at various stagesduring the purification process.

FIG. 15 shows the fold increase over control (background from MMP-2sensor).

FIGS. 16A-B show the luminescence of the MMP-2 protein using theSensoLyte assay, (FIG. 16A) and the fold-induction (FIG. 16B).

FIG. 17 shows cleavage of CBS-HT by Caspase-3 detected by SDS-PAGE gelanalysis.

FIGS. 18A-B show illustrations of the immobilization of CBS to aHaloLink resin (FIG. 18A) or a microtiter plate (FIG. 18B).

FIG. 19 shows the luminescence of protease biosensor expressed in acell-free environment.

FIG. 20 shows the luminescence of protease biosensor expressed in E.coli.

FIGS. 21A-B show SDS-PAGE analysis of samples labeled with CA-TAM (FIG.21A) and the luminescence of purified protease biosensor (FIG. 21B).

FIG. 22 shows the luminescence of the protease biosensor immobilized toa solid support.

DETAILED DESCRIPTION

Before any embodiments of the invention are explained in detail, it isto be understood that the invention is not limited in its application tothe details of construction and the arrangement of components set forthin the following description or illustrated in the following drawings.The invention is capable of other embodiments and of being practiced orof being carried out in various ways.

In the following description of the methods of the invention, processsteps are carried out at room temperature (about 22° C.) and atmosphericpressure unless otherwise specified. It also is specifically understoodthat any numerical range recited herein includes all values from thelower value to the upper value. For example, if a concentration range orbeneficial effect range is stated as 1% to 50%, it is intended thatvalues such as 2% to 40%, 10% to 30%, or 1% to 3%, etc. are expresslyenumerated in this specification. Similarly, if a sequence identityrange is given as between, e.g., 60% to <100%, it is intended that 65%,75%, 90%, etc. are expressly enumerated in this specification. These areonly examples of what is specifically intended, and all possiblenumerical values from the lowest value to the highest value areconsidered expressly stated in the application.

The term “thermostable luciferase” includes a luciferase that hasenhanced stability at a given temperature (e.g., 22° C.) compared to acorresponding wild-type luciferase. For the exemplary embodimentsdisclosed herein, the term “TL” is used to refer to a thermostablevariant of Ppe2, where Ppe2 is a luciferase from Photuris pennsylvanica.However, one skilled in the art would recognize that any thermostableluciferase could be used where TL is stated. For example, a luciferasefrom Photinus pyralis may be used, as well as luciferases from Luciolacruciata, Luciola lateralis, Pyrocoelia miyako, Lampyris noctiluca,Photuris pennsylvanica, Phengodes sp., Luciola mingrelica, and Photinuspyralis. (See Ye et al., Biochimica et Biophysica Acta, 1339:39-52(1997)).

The term “CP” refers to circularly-permuted. For example, “TL-CP” refersto a circularly-permuted thermostable variant of the Ppe2 luciferasefrom Photuris pennsylvanica. The term “DEVD:DD” refers to a linker,i.e., an amino acid sequence that connects the N- and C-terminals of acircularly-permuted luciferase, that contains the DEVD caspase 3/7recognition site and the three amino acids, GSL, that are on theC-terminal side of the DEVD caspase recognition site.

The term “biosensor” refers to an amino acid sequence containing asensor region which can interact with a target molecule. When the targetmolecule interacts with the sensor region, molecular properties of asystem are revealed.

The terms “Caspase-3/7 BioSensor” and “CBS” refers to a biosensorcomprising a thermostable variant of the Ppe2 luciferase from Photurispennsylvanica circularly-permuted with a caspase-3/7 recognition site,i.e., one containing the caspase-3/7 recognition site, DEVD, at thejunction between the modified TL fragments. For example,“TL-CP358-DEVD:DD” refers to a CBS circularly-permuted at position 358relative to SEQ ID NO:2 and comprises the DEVD:DD linker connecting theN- and C-terminal ends of the circularly-permuted thermostableluciferase. The term “CBS variant” refers to a CBS with one or moreamino acid substitutions relative to CBS.

The amino acid numbering used throughout this application to identifysubstituted residues is specified relative to the positions in thepolypeptide sequence of the wild-type Ppe2 luciferase from Photurispennsylvanica, i.e., SEQ ID NO:2, or the thermostable variant of thePpe2 luciferase from Photuris pennsylvanica polypeptide sequence, i.e.,SEQ ID NO:4. In addition, other mutants than that shown in SEQ ID NO:4can be used.

The term “target molecule” refers to a molecule of interest thatinteracts with the biosensor, e.g., a protease, a kinase, a G-proteincoupled receptor, cAMP, cGMP, enzyme cofactors, ions (e.g., calcium ion;hydrogen ion for use as a pH sensor), an antibody, a peptide, or a sugarthat causes the biosensor to reveal molecular properties of a system.

The term “identity” in the context of two or more nucleic acids orpolypeptide sequences, refers to two or more sequences or subsequencesthat are the same or have a specified percentage of amino acid residuesor nucleotides that are the same when compared and aligned for maximumcorrespondence over a comparison window or designated region as measuredusing any number of sequence comparison algorithms or by manualalignment and visual inspection. Methods of alignment of sequence forcomparison are well-known in the art.

The terms “cell,” “cell line,” and “host cell,” as used herein, are usedinterchangeably, and all such designations include progeny or potentialprogeny of these designations. The term “transformed cell” refers to acell into which (or into an ancestor of which) has been introduced anucleic acid molecule of the invention. Optionally, a nucleic acidmolecule of the invention may be introduced into a suitable cell line soas to create a stably-transfected cell line capable of producing theprotein or polypeptide encoded by the nucleic acid molecule of theinvention. Vectors, cells, and methods for constructing such cell linesare well known in the art. The words “transformants” or “transformedcells” include the primary transformed cells derived from the originallytransformed cell without regard to the number of transfers. All progenymay not be precisely identical in DNA content, due to deliberate orinadvertent mutations. Nonetheless, mutant progeny that have the samefunctionality as screened for in the originally transformed cell areincluded in the definition of transformants.

As used herein, the term “heterologous” nucleic acid sequence or proteinrefers to a sequence that, relative to a reference sequence, has adifferent source, e.g., originates from a foreign species, or, if fromthe same species, it may be substantially modified from the originalform. The term “homology” refers to a degree of complementarity betweentwo or more sequences. There may be partial homology or completehomology (i.e., identity).

The term “nucleic acid molecule,” “polynucleotide,” or “nucleic acidsequence” as used herein, refers to nucleic acid, DNA or RNA, thatcomprises coding sequences necessary for the production of a polypeptideor protein precursor. The encoded polypeptide may be a full-lengthpolypeptide, a fragment thereof (less than full-length), or a fusion ofeither the full-length polypeptide or fragment thereof with anotherpolypeptide, yielding a fusion polypeptide.

A polynucleotide encoding a protein or polypeptide means a nucleic acidsequence comprising the coding region of a gene, or in other words, thenucleic acid sequence encodes a gene product. The coding region may bepresent in a cDNA, genomic DNA or RNA form. When present in a DNA form,the oligonucleotide may be single stranded (i.e., the sense strand) ordouble stranded. Suitable control elements such as enhancers/promoters,splice junctions, polyadenylation signals, etc. may be placed in closeproximity to the coding region of the gene if needed to permit properinitiation of transcription and/or correct processing of the primary RNAtranscript. Other control or regulatory elements include, but are notlimited to, transcription factor binding sites, splicing signals,polyadenylation signals, termination signals and enhancer elements.

As used herein, “parental” refers to the starting amino acid ornucleotide sequence that is used to generate the variants with furthermanipulations of the present invention. For example a wild-type Photurispennsylvanica Ppe2 luciferase (SEQ ID NO:2), a thermostable variant ofthe Ppe2 luciferase from Photuris pennsylvanica, such as SEQ ID NO:4, ora circularly-permuted thermostable variant of the Ppe2 luciferase fromPhoturis pennsylvanica, such as SEQ ID NO:6, can be used as the startingsequence to generate the variants described in the present invention. Inaddition, other variants besides those shown in SEQ ID NOs:4 or 6 can beused as the parental sequence.

By “peptide,” “protein” and “polypeptide” is meant any chain of aminoacids, regardless of length or post-translational modification (e.g.,glycosylation or phosphorylation). The nucleic acid molecules of theinvention may also encode a variant of a naturally-occurring protein orpolypeptide fragment thereof, which has an amino acid sequence that isat least 60%, 70%, 80%, 85%, 90%, 95% or 99% identical to the amino acidsequence of the naturally-occurring (native or wild-type) protein fromwhich it is derived. For example, a coleopteran luciferase has at least60%, 70%, 80%, 85%, 90%, 95% or 99% amino acid sequence identity to SEQID NO:2; a firefly luciferase has at least 60%, 70%, 80%, 85%, 90%, 95%or 99% amino acid sequence identity to one of SEQ ID NO:2 or 4 or theluciferases on which SEQ ID NOs:6, 8, 10, 12, 14, 18, 20, 22, 58, 60,62, 64, 66, 68, 70, 72, or 74 are based.

As used herein, “pure” means an object species is the predominantspecies present (i.e., on a molar basis it is more abundant than anyother individual species in the composition), and in one embodiment asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50% (on a molar basis) of allmacromolecular species present. Generally, a “substantially pure”composition will comprise more than about 80% of all macromolecularspecies present in the composition, in one embodiment more than about85%, about 90%, about 95%, or about 99%. In one embodiment, the objectspecies is purified to essential homogeneity (contaminant species cannotbe detected in the composition by conventional detection methods)wherein the composition consists essentially of a single macromolecularspecies.

Nucleic acids are known to contain different types of mutations. A“substitution” refers to an alteration in the sequence of a nucleotideat one or more base position(s) from the parental sequence. Mutationsmay also refer to insertion or deletion of one or more bases, so thatthe nucleic acid sequence differs from a parental sequence (e.g., awild-type) or has a replacement stop codon.

The term “responsivity” refers to the alteration in luminescence, e.g.,increased or decreased luminescence, due to the interaction of thebiosensor with the target molecule.

As used herein, a “sample” may refer to a cell, an animal, cell lysate,or an in vitro transcription/translation mixture.

The term “vector” refers to nucleic acid molecules into which fragmentsof DNA may be inserted or cloned and can be used to transfer DNAsegment(s) into a cell and capable of replication in a cell. Vectors maybe derived from plasmids, bacteriophages, viruses, cosmids, and thelike.

The term “wild-type” as used herein, refers to a gene or gene productthat has the characteristics of that gene or gene product isolated froma naturally occurring source. The gene or gene product can be naturallyoccurring or synthetic. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designatedthe “wild-type” form of the gene. In contrast, the term “mutant” or“variant” refers to a gene or gene product that displays modificationsin sequence and/or functional properties (i.e., altered characteristics)when compared to the wild-type gene or gene product. It is noted thatnaturally-occurring and synthetic mutants can be isolated and areidentified by the fact that they have altered characteristics whencompared to the wild-type gene or gene product.

Luminescence refers to the light output of a luciferase polypeptideunder appropriate conditions, e.g., in the presence of a suitablesubstrate such as a luciferin. The light output may be measured as aninstantaneous or near-instantaneous measure of light output (which issometimes referred to as “T=0” luminescence or “flash”) upon start ofthe luminescence reaction, which may start upon addition of theluciferin substrate. The luminescence reaction in various embodiments iscarried out in a solution containing lysate, for example, from the cellsin a prokaryotic or eukaryotic expression system. In other embodiments,expression occurs in an in vitro system, or the luciferase protein issecreted into an extracellular medium, such that, in this latter case,it is not necessary to produce a lysate. In other embodiments, theluciferase is expressed in a whole cell(s) or in vivo, e.g., in animals.In some embodiments, the reaction is started by injecting appropriatematerials, e.g., luciferin, into a reaction chamber (e.g., a well of amultiwell plate such as a 96-well plate) containing the luciferaseprotein. The reaction chamber may be situated in a reading device whichcan measure the light output, e.g., using a luminometer orphotomultiplier. When the luciferase is expressed in whole cell(s) or inan animal, the reaction is started by the administration of a luciferasesubstrate, e.g., luciferin. For a whole cell(s), this administration mayinclude addition of the luciferase substrate into the cell media. Foranimals, administration of the luciferase substrate may includeinjection or oral administration, e.g., inclusion of the substrate intothe animal's food or water. The light output or luminescence may also bemeasured over time, for example in the same reaction chamber, cell(s) oranimal, for a period of seconds, minutes, hours, etc. The light outputor luminescence may be reported as the average over time, the half-lifeof decay of signal, the sum of the signal over a period of time, or asthe peak output. Luminescence can also be detected via imaging, e.g., invivo imaging.

Enhanced response includes the differential activity before and afterthe TL-CP biosensor interacts with a target molecule. The basal activityof the TL-CP biosensor is defined as the activity at assay time (0),before the biosensor interacts with a target molecule. The inducedactivity is defined as the activity at some later time (t) after theTL-CP biosensor has been interacted with a target molecule. The responseor fold increase in activity is the ratio of induced to basal activity.

Enhanced luminescence includes increased light output as determined bysuitable comparison of comparably-obtained measurements. As disclosedherein, one or more suitable amino acid substitutions to the TL-CPbiosensor sequence produce TL-CP biosensor polypeptides which exhibitenhanced luminescence. Changes in the nucleotide sequence from theparental thermostable luciferase nucleotide sequence may contribute toenhanced luminescence by leading to an amino acid substitution and/or byenhancing protein expression.

Enhanced signal stability includes an increase in how long the signalfrom a luciferase continues to luminescence, for example, as measured bythe half-life of decay of the signal in a time-course.

Enhanced protein stability includes increased thermostability (e.g.,stability at elevated temperatures) and chemical stability (e.g.,stability in the presence of denaturants such as detergents, includinge.g., Triton X-100).

Luciferase biosensors have been previously described, see e.g., U.S.Patent Publication No. 2005/0153310, the disclosure of which areincorporated by reference herein. The sensor regions are cloned into acircularly-permuted luciferase such that when the luciferase biosensorinteracts with a target molecule, an enhanced or increased luminescenceis generated relative to a luciferase biosensor which has not beencontact with a target molecule. Alternatively, the sensor regions arecloned into a circularly-permuted luciferase such that when theluciferase biosensor interacts with a target molecule, a decrease or noluminescence is generated relative to a luciferase biosensor which hasnot been in contact with a target molecule. The sensor regions may beuseful for detecting the activity of a protease, the binding of cyclicnucleotides such as cAMP and cGMP, the presence or concentration ofcalcium, other ions or antibodies, the presence or concentrations of oneor more G-protein coupled receptor ligands, a change in pH, the activityof a phosphatase or kinase or other enzymes, binding proteins ormolecules of interest such as a peptide or a sugar known to those ofskill in the art.

In one embodiment, a polynucleotide of the invention is optimized forexpression in a particular host. As used herein, optimization includescodon optimization as well as, in eukaryotic cells, introduction of aKozak sequence, and/or one or more introns. Thus, a nucleic acidmolecule may have a codon composition that differs from that of awild-type nucleic acid sequence encoding an unmodified luciferase atmore than 30%, 35%, 40% or more than 45%, e.g., 50%, 55%, 60% or more ofthe codons.

In one embodiment of the invention, the codons that are different arethose employed more frequently in a mammal, while in another embodimentthe codons that are different are those employed more frequently in aplant. A particular type of mammal, e.g., human, may have a differentset of preferred codons than another type of mammal. Likewise, aparticular type of plant may have a different set of preferred codonsthan another type of plant. In one embodiment of the invention, themajority of the codons which differ are ones that are preferred codonsin a desired host cell, as those optimized sequences can increase thestrength of the signal for luciferase. Preferred codons for mammals(e.g., humans) and plants are known to the art (e.g., Wada et al. NAR18: 2367 (1990); Murray et al. NAR 17: 477 (1989); WO 02/16944).

In one embodiment, the corresponding wild-type luciferase is from aColeopteran species, e.g., Luciola cruciata, Luciola lateralis,Pyrocoelia miyako, Lampyris noctiluca, Photuris pennsylvanica, Phengodessp., Luciola mingrelica, and Photinus pyralis. (See Ye et al.,Biochimica et Biophysica Acta, 1339:39-52 (1997)).

In one embodiment, the TL-CP biosensor contains the caspase-3recognition site comprising amino acids DEVD, at the junction betweenthe TL. Upon interaction with caspase-3, the TL-CP biosensor is cleavedat the recognition site allowing the two TL fragments to form a morefavorable, higher activity, conformation. In other embodiments, theTL-CP biosensor contains a recognition site for other proteases, e.g.,caspase-8 recognition site (LETDG; SEQ ID NO:15), TEV proteaserecognition site (ENLYFQS; SEQ ID NO:16) or MMP-2 recognition site(PLGMWSR; SEQ ID NO: 75).

The amino acid sequence of the modified TL-CP biosensor is differentthan the amino acid sequence of a corresponding unmodified TL-CPbiosensor (parental), e.g., a mutant luciferase with one or moresubstitutions in the luciferase sequences. In one embodiment, theluciferase sequences of the modified thermostable luciferase arecircularly-permuted relative to the amino acid sequence of acorresponding unmodified thermostable luciferase (parental luciferase)wherein the permutation is at a site (residue) or in a region that istolerant to modification.

In one embodiment, the TL-CP biosensor has one or more discrete(isolated) heterologous amino acid sequences, at least one of whichdirectly or indirectly interacts with a target molecule, and optionallymay include the deletion of one or more amino acids, e.g., at a site(s)or in a region(s) tolerant to modification including the N- and/orC-terminus of the unmodified thermostable luciferase, so long as theresulting TL-CP biosensor has bioluminescent activity before and/orafter the interaction with the target, e.g., bioluminescent activity isaltered after interaction with the target molecule, such as analteration in light intensity, color or kinetic profile.

In one embodiment, a TL-CP of the invention comprises an amino acidsequence which is circularly-permuted relative to the amino acidsequence of a corresponding thermostable luciferase, such as anunmodified thermostable luciferase, resulting in a new N- and C-terminusin the circularly-permuted thermostable luciferase, at least one ofwhich is at a site or in a region which is tolerant to modification, andis engineered to have functionality by introducing a sensor regioncomprising an amino acid sequence which directly or indirectly interactswith a target molecule. In another embodiment, the circularly-permutedthermostable luciferase includes other modifications, including but notlimited to, insertions and/or deletions internal to the N- or C-terminusof the circularly-permuted thermostable luciferase, for instance,another insertion and/or a deletion, e.g., at or near the N- andC-terminus of the corresponding unmodified thermostable luciferase suchas at residues corresponding to residues 1 to about 10 or about 30, orany integer in between, of the N-terminus and/or corresponding to thelast residue or about the last 30, e.g., last 15, or any integer inbetween 1 and 30, residues of the C-terminus of the correspondingunmodified thermostable luciferase.

In one embodiment, a thermostable beetle luciferase may becircularly-permuted at a residue, for instance, residue 7, 37, 47, 75,83, 107, 121, 144, 160, 174, 188, 198, 205, 225, 233, 242, 255, 268,308, 316, 358, 377, 403, 435, 490 or 540, or in a region correspondingto residue 2 to 12; residue 32 to 53, e.g., residue 32 to 43 or residue42 to 52; residue 70 to 88, e.g., residue 70 to 80 or residue 78 to 88;residue 102 to 126, e.g., residue 102 to 112 or residue 116 to 126;residue 139 to 165; residue 183 to 203; residue 220 to 247, e.g.,residue 228 to 238; residue 262 to 273; residue 303 to 313; residue 353to 408; residue 485 to 495; or residue 535 to 546 of a fireflyluciferase, such as one of SEQ ID NOs:2 or 4. The residue numbering isbased on that of an unmodified (native) firefly luciferase sequence.Corresponding positions may be identified by aligning luciferasesequences using, for instance, sequence alignment programs. Residues orregions in a luciferase tolerant to modification may be employed assites to circularly permute the luciferase or for an insertion.

In one embodiment, the invention provides a polynucleotide encoding abiosensor comprising a modified circularly-permuted thermostableluciferase and a linker. In one embodiment, the thermostable luciferaseis based on a version of Photuris pennsylvanica luciferase Ppe2 (SEQ IDNOs: 1 and 2) comprising amino acid substitutions which confer improvedproperties such as thermostability (SEQ ID NOs:3 and 4). The linkerlinks the C-terminal portion of the modified thermostable luciferase tothe N-terminal portion of the modified thermostable luciferase. Thelinker has a sensor region capable of interacting with a target moleculein a cell. The modified thermostable luciferase biosensor has anenhanced response after interaction of the biosensor with the targetrelative to an unmodified thermostable luciferase biosensor.

In one embodiment, the modified circularly-permuted thermostableluciferase biosensor has enhanced response after interaction with atarget molecule in cells. The modified circularly-permuted thermostableluciferase biosensor include a substitution of at least one amino acidcorresponding to positions 5, 17, 21, 23, 26, 39, 44, 51, 81, 101, 103,110, 114, 115, 119, 123, 126, 128, 133, 137, 186, 191, 192, 193, 196,208, 211, 214, 226, 228, 230, 233, 264, 273, 275, 286, 287, 294, 295,297, 302, 303, 304, 306, 308, 309, 313, 324, 329, 331, 343, 348, 353,364, 374, 385, 389, 409, 420, 426, 427, 428, 431, 449, 456, 460, 461,465, 466, 468, 471, 473, 482, 484, 485, 489, 493, 494, 497, 503, 507,509, 510, 513, 516, 517, 521, 522, 523, 526, 530, 533, 536, 537, 542, or543 of SEQ ID NO: 2, or combinations thereof.

In one embodiment, a TL-CP has a linker containing a sensor regionconnecting the N- and C-terminals of the thermostable luciferase, wherethe sensor region comprises an amino acid sequence, e.g., a proteaserecognition site or a kinase site, which directly interacts with atarget molecule, e.g., a protease or kinase.

In one embodiment, the amino acid sequence that interacts with thetarget molecule is flanked by at least one linker, e.g., flanked at eachend, such as a peptide linker, which linkers may be the same ordifferent, which optionally improve luminescence and/or response uponinteraction with a target molecule. In one embodiment, the amino acidsequence that interacts with the target molecule is flanked by at leastone linker at the N-terminus, which optionally improves luminescenceand/or response upon interaction with a target molecule. In oneembodiment, the linker has at least one of the following sequences:

(SEQ ID NO: 23) G S S G G S G G S G G G,  (SEQ ID NO: 24)G S S S D S D S S A G S,  (SEQ ID NO: 25) G S N D S S G G S E G G, (SEQ ID NO: 26) G S N G G F D S S E G G,  (SEQ ID NO: 27)G S I R W S G L S G G D,  (SEQ ID NO: 28) G S R G G S V Y S E G G, (SEQ ID NO: 29) G S S E G S S D F G G D,  (SEQ ID NO: 30)G S I V V S C S S E G G,  (SEQ ID NO: 31) G S N W D S G C S R E G, (SEQ ID NO: 32) G S N W D S G C S R E C,  (SEQ ID NO: 33)G S S G C T G D A G G S,  (SEQ ID NO: 34) G S N W D S G C S R Q C, (SEQ ID NO: 35) G S S/N S/D/G D/S/G S/F D/G S/G S A/E G S/G,(SEQ ID NO: 36) G S I/R/S R/G/E W/G S G/V/S L/Y/D S/F G/E G D/G,(SEQ ID NO: 37) G S I/N/S V/W/G V/D/C S/T C/G S/C/D S/A E/R/G G/E G/S,(SEQ ID NO: 38) G S I/S V/G/A V/G S/C G/D G/D/S S/A G/E G/E G/N,(SEQ ID NO: 39)G S I/N/S V/W/G/A V/D/C/G S/T/C C/G S/C/D S/A E/R/G G/E G/S,(SEQ ID NO: 40) G S I A G C G D A G E G,  (SEQ ID NO: 41)G S N W D S G C S R E,  (SEQ ID NO: 42) G S I A G C G D A G E G, (SEQ ID NO: 43) G S N W D S G C S R E G,  (SEQ ID NO: 44)N W D S G C S R E G,  or (SEQ ID NO: 45) I A G C G D A G E G. 

The “/” mark indicates that the amino acid before or after the “/” maybe used in that position. A linker employed in the biosensor of theinvention is an amino acid sequence, the presence of which in thebiosensor does not substantially decrease the activity of thatbiosensor, e.g., does not decrease the activity by more than 10-fold,such as by no more that 4-fold, or no more than 2-fold, relative to acorresponding biosensor that lacks the linker(s), and/or the presence ofthe linker employed in the biosensor of the invention increasesluminescence or response to interacting with its target, relative to acorresponding biosensor that lacks the linker(s) or a correspondingbiosensor having the linker(s) GSSGGSGGSGGG (SEQ ID NO:23), or relativeto both corresponding biosensors.

In one embodiment, a peptide linker of the invention is positionedN-terminal to a sensor region of the invention and is capable ofdirectly or indirectly interacting with a target molecule, e.g., amolecule to be detected. In one embodiment, a peptide linker of theinvention is positioned C-terminal to that peptide sequence in abiosensor of the invention. In one embodiment, a peptide linker of theinvention is positioned N-terminal and C-terminal to peptide sequencewhich is capable of directly or indirectly interacting with a targetmolecule to be detected.

In one embodiment, in the absence of a target molecule, the activity ofa modified circularly-permuted thermostable luciferase biosensor of theinvention is less than the activity of a corresponding parental(unmodified) circularly-permuted thermostable luciferase biosensor,e.g., the luminescence activity of the modified circularly-permutedthermostable luciferase biosensor is about 0.001%, 0.01%, 0.1%, 1%, 10%,20%, 50%, 70% or more, but less than 100% that of a correspondingparental (unmodified) circularly-permuted thermostable luciferasebiosensor, the activity of which circularly-permuted modifiedthermostable luciferase biosensor is optionally detectable. In anotherembodiment, in the absence of the target, the activity of a modifiedcircularly-permuted thermostable luciferase biosensor of the inventionis substantially the same or greater than the activity of a parental(unmodified) circularly-permuted thermostable luciferase biosensor,e.g., the luminescence activity of the modified circularly-permutedthermostable luciferase biosensor of the invention is about 1.5-fold,e.g., at least 2-, 3- or 5-fold or more, that of a parental (unmodified)circularly-permuted thermostable luciferase biosensor. In the presenceof the target molecule, the activity of the modified circularly-permutedthermostable luciferase biosensor of the invention is detectablyaltered. For instance, a detectable alteration in activity of a modifiedcircularly-permuted thermostable luciferase biosensor in the presence ofa target molecule is an alteration of at least 0.001%, 0.01%, 0.1%, 1%,10%, or 100%, and up to 2-fold, 4-fold, 10-fold, 100-fold, 1.000-fold,10.000-fold or more, relative to the activity of the modifiedcircularly-permuted thermostable luciferase biosensor in the absence ofthe target. Thus, the physical proximity of a target molecule whichinteracts with a sensor region present in the modifiedcircularly-permuted thermostable luciferase biosensor but not theparental (unmodified) circularly-permuted thermostable luciferasebiosensor, alters, e.g., decreases, eliminates or increases, theactivity of the modified circularly-permuted thermostable luciferasebiosensor. In one embodiment, the luminescent signal of a modifiedcircularly-permuted thermostable luciferase biosensor of the inventionin the presence of the target is increased relative to the luminescentsignal of a corresponding parental (unmodified) circularly-permutedthermostable luciferase biosensor luciferase in the presence of a targetmolecule.

The invention includes circularly-permuted biosensors, which luciferasesequence may include deletions of residues at the original (wild type)N- or C-termini, or both, e.g., deletion of 1 to 3 or more residues atthe N-terminus and 1 to 6 or more residues at the C-terminus, as well asa sensor region which interacts with a target molecule or are affectedby post-translational modifications (sensors). The luciferase sequencesof a modified circularly-permuted thermostable luciferase are the sameor are substantially the same as the amino acid sequence of anunmodified circularly-permuted thermostable luciferase biosensor. Apolypeptide or peptide having substantially the same sequence means thatan amino acid sequence is largely, but may not entirely be, the same andretains a functional activity of the sequence to which it is related. Ingeneral, two amino acid sequences are substantially the same orsubstantially homologous if they are at least 80% identical, e.g., haveat least 85%, 90%, 95%, 99% or more identity.

In one embodiment, the modification may be the introduction of arecognition site for a hydrolase including but not limited to proteases,peptidases, esterases (e.g., cholesterol esterase), phosphatases (e.g.,alkaline phosphatase) and the like. For instance, hydrolases include,but are not limited to, enzymes acting on peptide bonds (peptidehydrolases) such as aminopeptidases, dipeptidases, dipeptidyl-peptidasesand tripeptidyl-peptidases, peptidyl-dipeptidases, serine-typecarboxypeptidases, metallocarboxypeptidases, cysteine-typecarboxypeptidases, omega peptidases, serine endopeptidases, cysteineendopeptidases, aspartic endopeptidases, metalloendopeptidases,threonine endopeptidases, and endopeptidases of unknown catalyticmechanism. For example, a modified thermostable beetle luciferase of theinvention may comprise an enterokinase cleavage site, a caspase cleavagesite, a coronavirus protease site (STLQ-SGLRKMA; SEQ ID NO:46), a kinasesite, a HIV-1 protease site (SQNY-PIVQ or KAVRL-AEAMS; SEQ ID NO:47 andSEQ ID NO:48, respectively), a HCV protease site (AEDVVCC-SMSYS; SEQ IDNO:49) (see, e.g., Lee et al., 2003), a SARS virus protease site (e.g.,QTSITSAVLQSGFRKMAFPS; SEQ ID NO:50, or VRQCSGVTFQGKFKKIVKGT; SEQ IDNO:51), a Granzyme B site, a rhinovirus protease site, e.g., rhinovirus3C protease site, a prohormone convertase site, aninterleukin-16-converting enzyme site, a CMV assembling site, aleishmandysin site, B. anthracis lethal factor, a botulinum neurotoxinlight chain protease site, a beta-secretase site for amyloid precursorprotein (VKM-DAEF; SEQ ID NO:56), prostate specific antigen sequence, athrombin site, a renin and angiotensin-converting enzyme site, acathepsin D site, a matrix metalloproteinase site, a uPA site, a plasminsite, a binding site for a cation, such as a calcium binding domain, acalmodulin binding domain, a cellulose binding domain, a chitin bindingdomain, a maltose binding protein domain, or a biotin binding domain. Inanother embodiment, a modified thermostable beetle luciferase of theinvention may comprise a sequence recognized by a ligand such as anantibody or a metal such as calcium.

The invention also includes a stable cell line that expresses a modifiedcircularly-permuted thermostable luciferase biosensor, comprises anexpression cassette comprising a nucleic acid molecule encoding themodified circularly-permuted thermostable luciferase biosensor of theinvention, and/or comprises a vector (e.g., a plasmid, virus, ordefective viral particles) capable of expressing the nucleic acidmolecule of the invention in a host cell. In one embodiment, theexpression cassette comprises a promoter, e.g., a constitutive orregulatable promoter, operably linked to the nucleic acid sequence. Inone embodiment, the expression cassette contains an inducible promoter.Also provided is a host cell, e.g., a prokaryotic cell or an eukaryoticcell such as a plant or vertebrate cell, e.g., a mammalian cell,including but not limited to a human, non-human primate, canine, feline,bovine, equine, ovine or rodent (e.g., rabbit, rat, ferret or mouse)cell, which comprises the expression cassette or vector of theinvention, and a kit which comprises the nucleic acid molecule,expression cassette, vector, host cell or modified circularly-permutedthermostable luciferase biosensor of the invention.

For instance, a vector encoding a modified circularly-permutedthermostable luciferase biosensor is mixed with a sample, e.g., a cell,cell lysate, in vitro transcription/translation mixture, or supernatant,and the activity of the modified circularly-permuted thermostableluciferase biosensor in the sample detected or determined, e.g.,optionally at one or more time points, or relative to a control samplewithout the target or having a differing amount of the target. Analteration in luminescent activity in the sample, for instance, overtime, and/or relative to a control, e.g., a cell having a specifiedamount of a target molecule, indicates the presence or amount of thetarget molecule in the sample, or change in amount of the targetmolecule related to experimental condition. In one embodiment, a cell iscontacted with a vector comprising a promoter, e.g., a regulatable orconstitutive promoter, and a nucleic acid sequence encoding a modifiedcircularly-permuted thermostable luciferase of the invention whichcomprises a sensor region which interacts with a cyclic nucleotide. Inone embodiment, a transfected cell is cultured under conditions in whichthe promoter induces transient expression of the modifiedcircularly-permuted thermostable luciferase biosensor, and the presenceor amount of luminescence determined. In another embodiment, a modifiedcircularly-permuted thermostable luciferase biosensor of the inventioncomprising a sensor region which interacts with a target molecule and asample suspected of having the target molecule are mixed, and the amountof luminescence determined.

A modified circularly-permuted thermostable luciferase biosensor of theinvention may be employed in applications where unmodifiedcircularly-permuted thermostable luciferase biosensor cannot, such as,as a functional reporter to measure or detect various conditions and/ortarget molecules in a cell or in an animal, e.g., a mouse. For instance,a vector encoding a modified circularly-permuted thermostable luciferasebiosensor, or the modified circularly-permuted thermostable luciferasebiosensor, is introduced to a cell, an animal, cell lysate, in vitrotranscription/translation mixture, or supernatant, and the activity ofthe modified circularly-permuted thermostable luciferase biosensordetected or determined, e.g., at one or more time points and relative toa corresponding unmodified circularly-permuted thermostable luciferasebiosensor. An alteration in luminescent activity in the cell, an animal,cell lysate, in vitro transcription/translation mixture, or supernatantover time, and/or relative to a control, e.g., a cell having thecorresponding unmodified circularly-permuted thermostable luciferasebiosensor, indicates the presence of the protease. For instance, theinvention includes a method to detect a virus associated with severeacute respiratory syndrome. The method includes contacting a biological,e.g., a physiological tissue or fluid, sample with a modifiedcircularly-permuted thermostable luciferase biosensor. The biosensorcomprises an amino acid recognition sequence for a protease of thevirus. It is detected or determined whether the activity of the modifiedcircularly-permuted thermostable luciferase biosensor in the presence ofthe sample is altered, thereby indicating whether the sample containsthe virus.

In an aspect, the disclosure provides a method to detect the presence oractivity of a target molecule in a sample comprising contacting thesample with a modified circularly-permuted thermostable luciferasebiosensor and a substrate for the modified circularly-permutedthermostable luciferase and measuring luminescence. In embodiments, themodified circularly-permuted thermostable luciferase comprises a sensorregion for the target molecule. The sensor region may contain but is notlimited to a protease recognition site, a kinase recognition site, anantibody binding site, a metal binding site, an ion biding site, acyclic nucleotide binding site or a nucleotide binding site. Inembodiments, the method may detect the presence or activity of targetmolecule which is a protease, a kinase, an antibody, a metal, an ion, acyclic nucleotide or a nucleotide. In embodiments, the protease may be acaspase 3, caspase 8, TEV protease or MMP-2. In embodiments, the samplemay be a cell, an animal, cell lysate, or an in vitrotranscription/translation mixture. In embodiments, the method furthercomprises adding a test compound wherein the test compound may alter(e.g., decreases, eliminates, or increases) the activity of the targetmolecule. In embodiments, the substrate for the modifiedcircularly-permuted thermostable luciferase biosensor may be luciferinor a luciferin derivative.

The invention also provides a method of detecting the presence of amolecule of interest. For instance, a cell is contacted with a vectorcomprising a promoter, e.g., a regulatable promoter, and a nucleic acidsequence encoding a modified circularly-permuted thermostable luciferasebiosensor of the invention which comprises an insertion/sensor regionwhich interacts with the molecule of interest. In one embodiment, atransfected cell is cultured under conditions in which the promoterinduces transient expression of the modified circularly-permutedthermostable luciferase biosensor, and a detectable activity of themodified circularly-permuted thermostable luciferase biosensor isdetermined. In another embodiment, an animal, e.g., a mouse, iscontacted with a vector comprising a promoter, e.g., a regulatablepromoter, and a nucleic acid sequence encoding a modifiedcircularly-permuted thermostable luciferase biosensor of the inventionwhich comprises an insertion/sensor region which interacts with themolecule of interest or a transfected cell expressing the modifiedcircularly-permuted thermostable luciferase biosensor of the presentinvention. Detectable activity of the modified circularly-permutedthermostable luciferase biosensor is then determined.

The modified circularly-permuted thermostable luciferase biosensor ofthe invention comprises an amino acid sequence which interacts with atarget molecule, i.e., molecule of interest, or is otherwise sensitiveto conditions relative to the corresponding unmodifiedcircularly-permuted thermostable luciferase biosensor. One or moremutated polynucleotides are selected which encode mutated modifiedcircularly-permuted luciferase biosensors that have an alteredinteraction with the molecule of interest or altered activity undercertain conditions relative to the interaction or activity of themodified circularly-permuted luciferase biosensor. In anotherembodiment, the invention provides a method which includes contacting amodified circularly-permuted thermostable luciferase biosensor of theinvention with a library of molecules, and detecting or determiningwhether one or more molecules interacts with the sensor region in themodified circularly-permuted thermostable luciferase biosensor.

The invention also provides methods of screening for agents (“test”agents) capable of modulating the amount of the target molecule ormolecule of interest present in a sample. “Modulation” refers to thecapacity to either enhance or inhibit a functional property ofbiological activity or process (e.g., enzyme activity). Such enhancementor inhibition may be contingent on the occurrence of a specific event,such as activation of a signal transduction pathway, and/or may bemanifest only in particular cell types. A “modulator” refers to an agent(naturally occurring or non-naturally occurring), such as, for example,a biological macromolecule (e.g., nucleic acid, protein, non-peptide, ororganic molecule), small molecules, an extract made from biologicalmaterials such as bacteria, plants, fungi, or animal (particularlymammalian) cells or tissues, or any other agent. Modulators areevaluated for potential activity as inhibitors or activators (directlyor indirectly) of a biological process or processes (e.g., agonist,partial antagonist, partial agonist, or antagonist) by inclusion in thescreening assays described herein. The activities (or activity) of amodulator may be known, unknown or partially known. Such modulators canbe screened using the methods of the invention. The term “test agent” or“test compound” refers to an agent or compound to be tested by one ormore screening method(s) of the invention as a putative modulator.Usually, various predetermined concentrations are used for screeningsuch as 0.01 μM, 0.1 μM, 1.0 μM, and 10.0 μM. Controls can include themeasurement of a signal in the absence of the test agent or compound,comparison to an agent or compound known to modulate the target, orcomparison to a sample (e.g., a cell, tissue or organism) before, duringand/or after contacting with the test agent or compound.

In one embodiment, the method includes screening for agents or compoundsthat modulate protease activity. For example, in one embodiment, amethod of identifying an agent or compound capable of modulatingapoptosis is provided. Caspase family proteases have been associatedwith apoptosis. Thus, the method includes contacting a sample suspectedof containing a caspase-family protease with an agent or compoundsuspected of modulating the caspase activity, and a modifiedcircularly-permuted thermostable luciferase biosensor having a cleavagesite cleavable by the caspase. The activity of the modifiedcircularly-permuted thermostable luciferase biosensor is detected in thesample before and after contacting with the test agent or compound. Anincrease in activity after contacting with the agent is indicative of anagent or compound that inhibits apoptosis and a decrease is indicativeof an agent that activates apoptosis.

Accordingly, the invention provides a screening system useful foridentifying agents or compounds which modulate the cleavage ofrecognition sequence present in a modified circularly-permutedthermostable luciferase biosensor of the invention and detecting itsactivity. This allows one to rapidly screen for protease activitymodulators. Utilization of the screening system described hereinprovides a sensitive and rapid means to identify agents or compoundswhich modulate (e g, inhibit or activate) a protease, for example, acaspase family protease. In particular, the invention contemplatesmodified circularly-permuted thermostable luciferase biosensors in whichthe sensor region includes an amino acid sequence that is a cleavagesite for an enzyme of interest. Thus, when the molecule of interest is aprotease, the insertion comprises a peptide containing a cleavagerecognition sequence for the protease. A cleavage recognition sequencefor a protease is a specific amino acid sequence recognized by theprotease during proteolytic cleavage. Accordingly, the inventionprovides methods to determine the amount of a protease in a sample bycontacting the sample with a modified circularly-permuted thermostableluciferase biosensor of the invention comprising a sensor region for theprotease and measuring changes in luciferase activity. The modifiedcircularly-permuted thermostable luciferase biosensor of the inventioncan be used for, among other things, monitoring the activity of aprotease inside a cell or an animal that expresses the modifiedcircularly-permuted thermostable luciferase biosensor.

The assays of the invention can be used to screen drugs to identifyagents or compounds that alter the activity of a protease that cleavesthe modified circularly-permuted thermostable luciferase biosensor. Inone embodiment, the assay is performed on a sample in vitro containing aprotease. A sample containing a known amount of protease is mixed with amodified circularly-permuted thermostable luciferase biosensor of theinvention and with a test agent. The amount of the protease activity inthe sample is then determined as described above. The amount of activityper mole of protease in the presence of the test agent is compared withthe activity per mole of protease in the absence of the test agent. Adifference indicates that the test agent alters the activity of theprotease. Accordingly, the alterations may be an increase in proteaseactivity resulting in a decrease in modified circularly-permutedthermostable luciferase biosensor activity or a decrease in proteaseactivity corresponding to an increase or maintenance of modifiedcircularly-permuted thermostable luciferase biosensor activity.

In one embodiment, the ability of an agent to alter protease activity isdetermined. In this assay, cells are conditioned or contacted with anagent or compound suspected of modulating protease activity. The cell orcells in the culture are lysed and protease activity measured. Forexample, a lysed cell sample containing a known or unknown amount ofprotease is mixed with a modified circularly-permuted thermostableluciferase biosensor of the invention. The amount of the proteaseactivity in the sample is then determined as above by determining thedegree of modified circularly-permuted thermostable luciferase biosensoractivity in a control or non-treated sample and the treated lysedcellular sample. The activity or inhibition can be calculated based on aper microgram or milligram protein in the sample. Accordingly, themodulation in protease activity includes an increase in proteaseactivity resulting in a decrease in modified circularly-permutedthermostable luciferase biosensor activity or a decrease in proteaseactivity corresponding to an increase or maintenance of modifiedcircularly-permuted thermostable luciferase biosensor activity.Typically, the difference is calibrated against standard measurements toyield an absolute amount of protease activity. A test agent thatinhibits or blocks the activity or expression of the protease can bedetected by increased modified circularly-permuted thermostableluciferase biosensor activity in treated cells compared to untreatedcontrols.

In another embodiment, the ability of an agent or compound to alterprotease activity in vivo is determined. In an in vivo assay, cellstransfected, either transiently or stably, with an expression vectorencoding a modified circularly-permuted thermostable luciferasebiosensor of the invention are exposed to different amounts of the testagent or test compound, and the effect of the test agent or testcompound on luciferase activity in a cell can be determined. Typically,the difference is calibrated against standard measurements to yield anabsolute amount of protease activity. A test agent that inhibits orblocks the activity or expression of the protease can be detected byincreased modified circularly-permuted thermostable luciferase biosensoractivity in treated cells compared to untreated controls.

In another embodiment, the ability of an agent or compound to alterprotease activity in an animal is determined. In a whole animal assay,an animal, e.g., mouse, may be injected with cells that express amodified circularly-permuted thermostable luciferase biosensor of theinvention, and the animal exposed to different amounts of a test agentor test compound. In embodiments, cells that express a modifiedcircularly-permuted thermostable luciferase biosensor of the inventionmay be implanted in an animal. In embodiments, the substrate for themodified circularly-permuted thermostable luciferase is injected intothe animal. In embodiments, the substrate is injected into the cells ofthe animal. The effect of the test agent or test compound on luciferaseactivity in the animal can then be determined.

The disclosure also provides a method of immobilizing the modifiedcircularly-permuted thermostable luciferase biosensor to a solidsupport, e.g., a particle, resin, column, solid surface (e.g., plate,slide, or well bottom), etc. The immobilized biosensor can then be usedto detect the presence or activity of a molecule of interest. Inembodiments, the modified circularly-permuted thermostable luciferasebiosensor of the invention, either in purified form or expressed in celllysate, e.g., E. coli cell lysate, can be immobilized onto a solidsupport, e.g., resin or solid surface, and a molecule of interestdetected. The molecule of interest can be purified form of the moleculeor also be expressed in a cell lysate. Detectable activity of themodified circularly-permuted thermostable luciferase biosensor is thendetermined.

In an aspect, the disclosure provides a method to detect apoptosis in asample comprising contacting the sample with a modifiedcircularly-permuted thermostable luciferase biosensor and a substratefor the modified circularly-permuted thermostable luciferase anddetecting luminescence in the sample. In embodiments, the modifiedcircularly-permuted thermostable luciferase biosensor contains a sensorregion for a molecule involved in apoptosis.

The materials and composition for use in the assay of the invention areideally suited for the preparation of a kit. Such a kit may comprise acarrier means containing one or more container means such as vials,tubes, and the like, each of the containers means comprising one of theseparate elements to be used in the method. One of the containerscomprises a modified circularly-permuted thermostable luciferasebiosensor or polynucleotide (e.g., in the form of a vector) of theinvention. A second container may contain a substrate for the modifiedcircularly-permuted thermostable luciferase biosensor.

The invention will be further described by the following non-limitingexamples.

EXAMPLES Example I Generation of a Modified Thermostable LuciferaseBiosensor with Increased Responsivity in Cells

The Caspase-3 BioSensor (CBS) is a thermostable Photuris pennsylvanicaluciferase (TL), circularly-permuted (CP) at amino acid 358, with acaspase-3 recognition site, i.e., one containing the caspase-3recognition site comprising amino acids DEVD, at the junction betweenthe TL fragments. The specific CBS that was used as the startingtemplate is termed TL-CP358-DEVD:DD. The amino acid sequence of this CBScan be represented as: M/TL residues 358-544/SDEVDGSL/TL residues4-354/V (SEQ ID NO:6). The amino acid positions in the CP TL correlateto those of the non-CP TL sequence (provided in the attached appendix).Upon treatment with caspase-3, CBS is cleaved at the recognition siteallowing the two TL fragments to form a more favorable, higher activity,conformation.

The utility of CBS is the differential activity before and aftercleavage by caspase-3. The basal activity of CBS is defined as theactivity at assay time (0), before caspase-3 has had time to cleave atthe recognition site. The induced activity is defined as the activity atsome later time (t) after CBS has been cleaved by caspase-3. Theresponse or fold increase in activity, is the ratio of induced to basalactivity. Substitutions in TL-CP358-DEVD:DD were generated to developCBS variants with enhanced responsivity to induction using theerror-prone, mutagenic PCR-based system GeneMorph II (Stratagene;Daugherty, PNAS USA 97(5):2029 (2000)), according to manufacturer'sinstructions.

The resulting library was expressed in E. coli and screened forluciferase activity with and without pre-treatment with recombinantcaspase-3 (data not shown). CBS variants having the best signal andresponse characteristics were then evaluated in HEK293 cells by kineticassay measuring the response to TNF-α-related apoptosis inducing ligand(TRAIL) treatment (Wiley, S. R. et al., Immunity 3:673 (1995); Niles, A.L. et al., Meth. Mol. Biol. 414:137 (2008)). TRAIL induces apoptosis viaactivation of the death receptor to form active caspase-8, which in turnactivates procaspase 3 to produce caspase-3. The appearance of activecaspase-3 should be accompanied by an increase in luminescence as theCBS variants are cleaved and activated. Briefly, HEK293 cells, plated at15,000 cells/well in a 96-well plate, were transiently transfected usingTranslT-LTI (Mims Bio) with plasmid DNAs encoding various CBS variantswith amino acid substitutions in TL-CP358-DEVD:DD. The same plasmidsalso carried a gene for constitutive expression of Renilla luciferase toact as a transfection control. Cells were pretreated with 2 mM luciferinfor 2 hrs at 37° C. Cells were treated with 1 μg/mL TRAIL and assayedfor 10 hrs at 37° C. Luminescence was monitored continuously over time(Luminometer: Varioskan Flash (Thermo) 1 sec integration time). Cells inreplicate wells were lysed, at the time of TRAIL addition, i.e., time(0) and Renilla luciferase activity was measured. All biosensor data wasthen normalized for transfection efficiency using Renilla luciferaseluminescence (Dual-GloAssay System; Promega Corporation).

Exemplary CBS variants include, but are not limited to those listed inTable 1. Table 1 lists the variants of TL-CP358-DEVD:DD, identified byclone name, showing improved response to TRAIL treatment. Improvementslisted in Table 1 are normalized to the parental TL-CP358-DEVD:DD CBS.“BASAL” represents the normalized biosensor luminescence at TRAILaddition, i.e., time (0), “INDUCED” represents the normalized biosensorluminescence at roughly 10 hrs after TRAIL addition, and “RESPONSE”represents the fold-induction, i.e., the ratio of INDUCED to BASALactivity.

Standard sequencing techniques known in the art were used to identifythe amino acid substitution in each clone (see Table 1). The amino acidposition is based on parental TL, e.g., Pro at position 2 of thevariants=TL 358; the residues to the N-terminus of the DEVD thereforerepresent TL residues 358-544(Gly); the residues to the C-terminus ofthe DEVD represent TL residues 4(Lys)-354(Gly) (See FIG. 1 forexamples). Each amino acid substitution is indicated by the positioncorresponding to the amino acid position in the parental TL, not theTL-CP358-DEVD:DD sequence, whereby the first letter following thenumerical position represents the corresponding amino acid in parentalTL. If the amino acid is substituted with another amino acid, the secondletter represents the amino acid substitution. If the amino acid issubstituted with a stop codon, the substitution is indicated by “STOP.”

TABLE 1 Summary of the fold improvement in responsivity of CBS variantsover the corresponding TL-CP358-DEVD:DD. IMPROVEMENT OVERTL-CP358-DEVD:DD CLONE BASAL INDUCED RESPONSE mut#1 mut#2 mut#3 mut#4mut#5 mut#6 01:E-12 0.35 0.71 2.04 021AD 01:G-12 0.00 0.00 2.11 044AD01:G-03 0.47 0.59 1.26 128SP 01:E-11 0.4 0.6 1.3 193SP 09:A-11 0.69 1.171.69 273LQ 01:H-06 0.22 0.39 1.77 275SP 13:F-03 0.30 1.02 3.41 503DG03:E-12 0.44 0.50 1.16 286LH 01:C-04 0.15 0.65 4.20 294LH 05:C-07 0.160.20 1.30 294LP 14:A-04 0.34 0.95 2.77 297SG 12:B-10 1.15 1.34 1.16297SI 13:B-03 0.74 1.45 1.95 329RW 14:F-06 0.20 1.12 5.74 409AV 05:C-020.19 0.38 2.02 461PL 06:A-12 0.04 0.14 3.26 465DV 01:D-02 0.1 0.6 5.3471IT 14:G-12 0.11 0.30 2.69 482AS 06:G-09 0.55 1.30 2.69 482AV 07:C-060.16 0.61 3.79 485VE 05:B-06 0.05 0.20 4.06 497VA 07:B-02 0.5 1.0 1.8503SG 01:A-05 0.2 0.6 3.6 507TI 05:D-11 0.04 0.16 3.55 522PS 02:B-090.25 0.55 2.39 526TS 06:D-04 0.18 0.45 2.42 530DV 01:F-02 0.58 0.67 1.15543NS 05:E-06 0.12 0.41 3.51 017EG 513GA 09:D-10 0.19 0.43 2.21 026FY530DG 13:B-04 0.51 1.26 2.48 051LS 193SP 01:H-08 0.34 0.67 1.96 110EV304DA 11:A-11 0.71 1.16 1.63 115HY 521IV 05:G-10 0.43 0.82 1.92 123RH303YD 04:A-05 0.21 0.45 2.20 128SP 523KQ 12:D-08 330.89 764.74 2.31192AT 389LS 05:D-06 0.55 1.03 1.87 193SP 114IK 15:E-10 0.06 0.17 2.73196FY 530DG 11:F-07 0.23 0.69 2.96 208MK 466AG 04:H-08 0.06 0.17 3.03226FY 509KT 07:C-02 0.29 0.78 2.66 264MT 303YN 01:E-04 0.22 0.57 2.63294LH 304DE 13:C-09 0.55 1.36 2.46 294LH 308LI 04:H-07 0.14 0.25 1.80294LP 510WR 14:G-10 0.23 0.50 2.17 302KE 530DV 01:B-08 0.07 0.15 2.20308LS 343E(STOP) 13:G-12 0.37 1.06 2.89 309KN 331KI 16:D-12 0.2 0.6 2.4329RQ 530DV 05:A-07 0.29 0.61 2.10 353K(STOP) 530DA 09:E-03 0.54 1.262.33 364IM 530DA 18:C-05 0.07 0.29 4.24 374DY 431FL 13:A-05 0.09 0.182.67 374DY 431FS 08:A-12 0.24 0.75 3.13 385EG 465DG 01:C-11 0.01 0.108.50 420GR 489GR 04:A-08 0.01 0.03 3.92 468VI 484VL 04:G-10 0.17 0.533.14 484VI 516KN 04:D-01 0.66 0.79 1.21 543NS 494EV 04:E-11 0.14 0.423.07 081SC 374DV 517FC 01:H-11 0.03 0.07 2.15 101VA 286LP 364IL 05:C-060.21 0.41 2.01 119IN 294LH 542TP 04:B-09 0.10 0.16 1.66 128SP 211HL287VA 11:D-05 0.20 0.46 2.25 137NY 493NY 507TI 10:D-04 0.17 0.41 2.33196FY 228NT 530DG 11:B-03 0.04 0.19 4.57 208ML 230IT 273LP 07:B-04 0.240.61 2.54 295AV 449AT 537ML 04:C-03 0.2 0.7 2.9 523KI 533VA 536QR10:D-11 0.51 1.31 2.58 005ND 133QL 228NT 294LH 15:D-11 0.27 1.00 3.64021AS 426DN 428DG 526TS 11:A-06 0.23 0.85 3.73 039IT 214IV 348VA 507TI14:H-06 0.16 0.69 4.24 186NI 233TM 427ND 465DG 05:F-11 0.94 1.50 1.58103PS 191VA 306SP 313ST 473DV 11:B-10 0.53 1.19 2.25 126FC 466AV 471IM536QR 543NK 10:D-03 0.34 1.06 3.09 023EG 228NT 309KE 324EG 456IV 460HL

Example II Evaluation of Specific Combinations of Mutations inThermostable Luciferase Caspase-3 Biosensors

Additional CBS variants were generated using the oligo-basedsite-directed mutagenesis kit Quik Change (Stratagene; Kunkel, PNAS USA82(2):488 (1985)), according to the manufacturer's instructions. Theamino acid substitutions identified in those variants from Example Iwith the most improved response, specifically clones 12:B-10, 01:A-05,04:C-03, 01:E-11, 16:D-12, 01:D-02 and 07:B-02, were combined andevaluated in HEK293 cells as in Example I. The amino acid substitutionsused to generate the additional CBS variants were 193SP, 297SI, 329RQ,471IT, 503SG, 507TI, 523KI, 533VA, and 536QR corresponding to SEQ IDNO:2. Exemplary CBS variants include, but are not limited to, thoselisted in Table 2. Table 2 identifies the clone (“NEW #”), the aminoacid substitutions found in the clone indicated by an X in the columnwhich indicate the amino acid substitution, 193SP, 297SI, 329RQ, 471IT,503SG, 507TI, 523KI, 533VA, and 536QR, the improvement in Basal, Inducedand Response over the corresponding starting TL-CP358-DEVD:DD.

TABLE 2 Summary of the fold improvement in responsivity of CBS variantswith specific amino acid substitution combinations over thecorresponding TL-CP358-DEVD:DD. IMPROVEMENT OVER TL-CP358-DEVD:DD NEW #193SP 297SI 329RQ 471IT 503SG 507TI 523KI 533VA 536QR BASAL INDUCEDRESPONSE FC7: 02 X X X X X X 0.023 0.070 3.112 FC7: 05 X X X X X X 0.0280.089 3.197 FC7: 06 X X X X X X 0.007 0.016 2.444 FC7: 07 X X X X X X0.005 0.008 1.483 FC7: 08 X X X X X 0.007 0.009 1.365 FC7: 12 X X X X X0.077 0.168 2.186 FC7: 15 X X X X X 0.038 0.048 1.253 FC7: 16 X X X X X0.010 0.019 2.014 FC7: 17 X X X X X 0.060 0.153 2.543 FC7: 18 X X X X X0.070 0.168 2.411 FC7: 19 X X X X X 0.161 0.353 2.194 FC7: 21 X X X X X0.123 0.235 1.920 FC7: 22 X X X X X 0.044 0.143 3.263 FC7: 24 X X X X0.035 0.191 5.480 FC7: 26 X X X X 0.065 0.186 2.843 FC7: 27 X X X X0.033 0.069 2.093 FC7: 28 X X X X 0.019 0.044 2.400 FC7: 30 X X X X0.110 0.288 2.614 FC7: 31 X X X X 0.241 0.496 2.053 FC7: 32 X X X X0.090 0.225 2.485 FC7: 33 X X X X 0.070 0.344 4.886 FC7: 36 X X X X0.218 0.390 1.789 FC7: 37 X X X X 0.435 0.775 1.781 FC7: 39 X X X X0.147 0.294 2.000 FC7: 40 X X X X 0.273 0.477 1.749 FC7: 41 X X X X0.186 0.573 3.081 FC7: 42 X X X X 0.131 0.235 1.803 FC7: 43 X X X 0.0280.189 6.735 FC7: 44 X X X 0.015 0.021 1.390 FC7: 45 X X X 0.184 0.5593.041 FC7: 46 X X X 0.125 0.349 2.782 FC7: 47 X X X 0.141 0.392 2.782FC7: 49 X X X 0.167 0.852 5.115 FC7: 50 X X X 0.192 0.724 3.765 FC7: 52X X X 0.178 0.369 2.077 FC7: 54 X X X 0.345 0.728 2.109 FC7: 55 X X X0.356 0.460 1.292 FC7: 56 X X X 0.644 0.818 1.271 FC7: 58 X X 0.1100.347 3.153 FC7: 59 X X 0.198 0.704 3.549 FC7: 60 X X 0.145 0.497 3.429FC7: 61 X X 0.505 0.926 1.833 FC7: 62 X X 0.460 0.955 2.078 FC7: 63 X X0.407 0.694 1.705 FC7: 64 X X X X 0.213 0.669 3.139 FC7: 65 X X X 0.0480.091 1.882

Many of the combinations of substitutions tested demonstrated increasedresponsivity as compared to the parental TL-CP358-DEVD:DD biosensor orthe variants disclosed in Table 1. Four CBS variants, namely 01:A-05,FC7:24, FC7:43 and FC7:49, were of particular interest, (see FIG. 1 andTable 3). FIG. 1 shows the position of the four amino acidsubstitutions, 147IT, S503G, T507I, and S193P, incorporated into thesevariants in the parental TL-CP358-DEVD:DD sequence and the positionscorrected for the circular permutation sites (see also Table 3). The topcartoon in FIG. 1 indicates the location of the substitutions based onsequential numbering of the primary amino acid sequence. The bottomcartoon in FIG. 1 indicates the codon designations based on the parentalTL-CP358-DEVD:DD. The nucleotide changes are as follows: 471: ata>aca;503: agt>ggt; 507: aca>ata; 193: tcg>ccg.

TABLE 3 Summary of amino acid substitutions found in clones 01:1-05,FC7:24, FC7:43 and FC7:43. Clone Substitution(s) 01:A-05 T507I FC7:24I471T S503G T507I S193P FC7:43 I471T S503G T507I FC7:49 S503G T507IS193P

The response to TRAIL in live cells in the improved CBS variants01:A-05, FC7:24, FC7:43 and FC7:49 (“1A5”, “24”, “43”, and “49”,respectively) was compared to the parental TL-CP358-DEVD:DD (“TL-CP”) inFIGS. 2A-B and 3A-B over a 10 hr time period. Variant 01:A-05 had 2times and about 4.8 times greater RESPONSE after 2 and 10 hrs TRAILtreatment, respectively, compared with TL-CP358-DEVD:DD (FIGS. 2A and2B). After 2 hrs, variants FC7:24 and FC7:49 had about 2 times greaterresponse than TL-CP358-DEVD:DD and variant 43 (FIGS. 3A and 3B). After10 hrs, variants FC7:24 and FC7:49 had about 3.2-3.7 times greaterresponse than TL-CP358-DEVD:DD (FIGS. 3A and 3B), while variant FC7:43had about 2.2 times greater response. These data demonstrates that CBSbiosensors can be generated to have improved response by incorporatingone or more of these four amino acid substitutions, 1471T, 5503G, T5071,and S193P.

Example III

Additional CBS variants were generated to have different linkersequences, such as SSDEVDGSSG (SEQ ID NO:52), SSGSDEVDGSLSSG (SEQ IDNO:53), SDEVDGSL (SEQ ID NO:54), or DEVDG (SEQ ID NO:55). The CBSvariants were evaluated in HEK293 cells as in Example I. Exemplary CBSvariants include, but are not limited to, those listed in FIG. 4. Allbiosensor data was then normalized for transfection efficiency usingRenilla luciferase luminescence as in Example I. FIG. 4 identifies theclone by the linker sequence it contains (“Linker”) and shows theluminescence in RLUs at TRAIL addition, i.e., time (0), (“Basal (t=0)”),at roughly 10 hrs after TRAIL addition (“Induced (10 h)”) and thefold-induction, i.e., the ration of Induced to Basal Activity (“Response(10 h)”). The common linker clone between the two experiments is #2(i.e., SSGSDEVDGSLSSG). The difference in the numbers is typicalvariation between experiments. Linker #3 is the same linker found in theclone referred to as “TL-CP358-DEVD:DD.”

Example IV Evaluation of the Mutant Thermostable Luciferase Biosensorsto Detect Caspase-8

To evaluate whether the mutant thermostable luciferase biosensors of thepresent invention can be used to detect Caspase-8 activity in cells,biosensors were generated that contained the Caspase-8 cleavage site,LETDG (SEQ ID NO:15). Two different biosensors, TL-CP358-Caspase-8 andTL-CP233-Caspase-8 were used. As controls, the firefly (Photinuspyralis; Ppy) luciferase biosensors FF-CP234-Caspase-8 (M/Ppy residues234-544/LETDG /Ppy residues 4-230N), FF-CP359-Caspase-8 (M/Ppy residues359-544/LETDG/Ppy residues 4-355/V), and TL-CP358-DEVD. Table 4 providessequence details of the biosensors.

TABLE 4 Caspase Cleavage Construct site with linker Luciferase FragmentsTL-CP358-Caspase 8 GSSLETDSSG TL Ppe 358-543 and 4-354(SEQ ID NOs: 59 and 60) (SEQ ID NO: 76) TL-CP233-Caspase 8 GSSLETDSSGTL Ppe 233-543 and 4-232 (SEQ ID NOs: 57 and 58) (SEQ ID NO: 76)FF-CP234-caspase-8 GSSLETDSSG Ppy 234-544 and 4-233(SEQ ID NOs: 21 and 22) (SEQ ID NO: 76) FF-CP359-caspase-8 GSSLETDSSGPpy 359-544 and 4-355 (SEQ ID NOs: 19 and 20) (SEQ ID NO: 76)TL-CP358-DEVD GSSDEVDSSG TL Ppe 358-543 and 4-354 (SEQ ID NOs: 5 and 6)(SEQ ID NO: 77)

All biosensors were transfected into HeLa cells. Cells were plated at a(10,000/well) into a 96-well tissue culture plate. Biosensor DNA wasprepared for transfection into the cells as described in Table 4. Thirty10 μL reactions were set up for each biosensor. TransIT® LTI (LTI; Mims)transfection master mix was prepared by mixing 1650 μL DMEM with 49.5 μLLTI. The master mix was incubated for 15 min at room temperature. 300 μLof the master mix was then added to each biosensor DNA (enough for 30reactions; 0.1 μg/reaction) and incubated for another 15 min at roomtemperature (Table 5). 10 μL of the biosensor DNA-transfection mastermix solution was added to the cells in the appropriate wells. The cellswere then incubated overnight at 37° C., 5% CO₂.

TABLE 5 FF-CP234- FF-CP359- TL-CP233- TL-CP358- caspase-8 caspase-8caspase-8 caspase-8 concentration 0.309 0.363 0.327 0.348 DNA amount for9.71 8.26 9.17 8.62 30 reactions (0.1 μg/well) volume of 300 300 300 300DMEM per 30 reactions (10 μL) μL of Mirus 9 9 9 9 LT1 per 30 reactions

After overnight incubation, the media was removed from the cells andreplaced with CO₂ Independent Media (Invitrogen Cat. No. 18045088) with2 mM Luciferin EF (Promega Cat. No. E6551). Cells were pre-equilibratedwith Luciferin EF for 2 hrs in a Varioskan luminometer withbioluminescence readings taken every 20 min. Following incubation, thecells were either induced with 1 μg/mL TRAIL in CO₂ IndependentMedia+10% Fetal Bovine Serum (FBS) or no compound (control; media+10%FBS only). The cells were again incubated at 37° C. in a Varioskanluminometer for 500+ min with bioluminescence measured every 20 min.

FIGS. 5-8 demonstrate at TL-CP233-Caspase 8 and TL-CP358-Caspase 8biosensors can detect Caspase 8 activation by TRAIL. FIGS. 5 and 7identify the kinetic profiles of Caspase 8 activation by TRAIL over timeat 37° C. FIGS. 6 and 8 identify the fold response of Caspase 8activation by TRAIL over time. Fold induction of activation wascalculated by dividing the signal of samples with TRAIL over the signalswithout trail at a given time point. FIGS. 7 and 8 show Caspase 3induction by Trail as measured by TL-CP358-DEVD as well as Caspase 8induction.

Example V Activation of TEV Protease Mutant Thermostable LuciferaseBiosensor

To evaluate whether the mutant thermostable luciferase biosensors of thepresent invention can detect TEV protease activity in cells, theTL-CP233 biosensor, TL-CP233-TEV, containing the TEV protease cleavagesite GSS-ENLYFQS-SSG (SEQ ID NO:78) was generated. TL-CP233-TEV has anamino acid sequence that can be represented as: M/TL residues233-544/GSS-ENLYFQS-SSG TL residues 4-233/V (SEQ ID NOs:61 and 62). Ascontrols, the firefly (Photinus pyralis; Ppy) luciferase biosensorsFF—CP235-TEV (M/Ppy residues 234-544/GSS-ENLYFQS-SSG/Ppy residues4-233/V; SEQ ID NOs:63 and 64), FF-CP269-TEV (M/Ppy residues269-544/GSS-ENLYFQS-SSG/Ppy residues 4-268/V; SEQ ID NOs:65 and 66), andFF—CP359-TEV (M/Ppy residues 359-544/GSS-ENLYFQS-SSG /Ppy residues4-355/V; SEQ ID NOs:67 and 68) were used. For all transfections, TEVprotease (Genbank accession no. BFB754) constitutively expressed from aCMV promoter was transfected (pF9a-BFB754). This construct alsoco-expresses Renilla luciferase for use as a transfection efficiencycontrol.

Each of the biosensors and TEV protease constructs were transfected inChinese Hamster Ovary (CHO) cells. Cells were plated at (15,000 cellsper well) into a 96-well tissue culture plate. The transfection solutionwas prepared according to Table 6. Each sensor was co-transfected witheither the TEV protease or a carrier vector (pF9a-null).

TABLE 6 Amount for 60 wells of a 96-well plate DNA (600 μL of media plus18 μL Mirus LT1 plus DNA) concentration 0.202 0.177 0.38 0.342 0.2610.214 0.214 construct pF9a-TEV pF9a Null FF-CP233- FF-CP268- FF-CP358-TL-CP233- Read protease TEV TEV TEV TEV Through Amount per Tfx (μg) 1.21.2 5.8 5.8 5.8 5.8 5.8 Amount per Tfx (μL) 5.9 6.8 15.3 17.0 22.2 27.127.1

Cells were incubated overnight for 24 hrs at 37° C., 5% CO₂. Afterovernight incubation, cells were equilibrated with media and 5 mMLuciferin EF for 2 hrs. Bioluminescence was then measured at 37° C. in aVarioskan luminometer. Results were normalized to Renilla to control fortransfection efficiencies.

The Biosensor without the TEV recognition sequence (FF—CP233-Readthrough; FF-CP233-RT) is not activated by TEV protease while the otherbiosensors were activated by TEV protease (FIG. 9).

Example VI Molecular Imaging of Apoptosis in Glioma Cells

To demonstrate that the mutant thermostable biosensors of the presentinvention can be used to detect cell death in cells, the thermostablecaspase 3 biosensors, TL-CP233-Caspase 3 (“233”; SEQ ID NOs:17 and 18),TL-CP358-Caspase 3 (“358V2”; SEQ ID NOs:5 and 6) and the mutantthermostable caspase 3 biosensors 1A5 (“358V3”; SEQ ID NOs:7 and 8), 24(“358V4”; SEQ ID NOs:9 and 10), 43 (“358V5”; SEQ ID NOs:11 and 12) and49 (“358V6”; SEQ ID NOs:13 and 14) were stably expressed in the gliomacell line D54-MG, the cells treated with TRAIL and bioluminescencemeasured to detect caspase 3 activity.

To derive cells stably expressing the thermostable biosensors, D54-MGcells were transfected with the biosensors. The biosensors weresubcloned into pEF vector containing a neomycin resistance gene(Invitrogen) via PCR amplification and inserted into the multiplecloning site at the SalI and EcoRI restriction sites. Transfections wereperformed in 6-well tissue culture dishes using 3 μL Fugene 6transfection reagent (Roche) and 1 μg plasmid DNA. Cells were placed inRPMI media (Gibco) containing 10% FBS (Gibco), Pen/Strep Glutamine(100×; Gibco) and 200 μg/mL geneticin (G418) for 48 hrs. Single cloneswere selected approximately 10 days after transfection using standardtechniques known in the art. Briefly, the media was removed from thecells, and the cells were gently washed with PBS. Round filter paperswere soaked in trypsin and placed on a single colony. The filter paper,which contained the attached cells, was removed and placed into a24-well tissue culture dish. Each individual clone was testedapproximately 2-3 weeks after selection for reporter expression byWestern blotting using a luciferase antibody (Promega; Cat. No. G7451)and bioluminescence (100 μg/mL D-Luciferin reconstituted in PBS was adddirectly to the media and detected). Clones with similar bioluminescentactivity (highest fold induction) and reporter expression (determined byWestern blot) were selected for use in detecting cell death.

To detect cell death, the stable D54-MG cells were seeded at 10,000cells/well into a 96-well assay plate and allowed to incubate for 24 hrsat 37° C., 5% CO₂. After overnight incubation, the cells were treatedwith 200 ng/mL TRAIL and 100 μg/mL D-luciferin (Promega). Live cellbioluminescence was imaged at 2, 4, and 6 hrs. Photon counts were takenat the different time points pre- and post-TRAIL treatment using theEnvision luminometer (Perkin Elmer). Reporter expression andTRAIL-induced apoptosis was further detected by Western blotting againstluciferase and Caspase-3.

FIG. 10 demonstrates that upon treatment with TRAIL, D54-MG cells stablyexpressing the various thermostable biosensors resulted in a 100-200fold induction in bioluminescent activity. D54-MG cells expressingdifferent versions of the thermostable biosensors were untreated ortreated with 200 ng/mL TRAIL and imaged at indicated time points; photoncounts/sec were recorded at the indicated time points (FIG. 10A). Thefold induction of D54-MG cells expressing different versions of thethermostable biosensors untreated or treated with 200 ng/mL TRAIL werecalculated by normalizing the values (photons/sec) to baseline (time 0hr) (FIG. 10B). The average photon counts/sec at baseline, 2 hrs, 4 hrsand 6 hrs post treatment in addition to fold changes achieved withdifferent biosensor versions are depicted in FIG. 10C. FIG. 10D showsthe detection of reporter expression and TRAIL induced apoptosis byWestern blotting against luciferase and Caspase-3.

Example VII Mutant Thermostable Biosensors Use

To demonstrate the use of the mutant thermostable biosensors to detectcell death in vivo, D54-MG cells stably expressing eitherTL-CP233-Caspase 3 (“233”; SEQ ID NOs: 17 and 18), TL-CP358-Caspase 3(“358V2”; SEQ ID NOs:5 and 6), 1A5 (“358V3”; “3-S”; SEQ ID NOs:7 and 8),43 (“358V5”; “5—R”; SEQ ID NOs:11 and 12) or 49 (“358V6”; “6-A”; SEQ IDNOs:13 and 14) were implanted into nude mice.

To establish a flank xenograft mouse model, 2×10⁶ D54-MG cells stablyexpressing one of the biosensors listed above (as described in ExampleVI) were implanted subcutaneously into nude mice. Treatment with 8 mg/kgTRAIL started when tumors reached ˜100 mm³ as assayed by electronicdigital caliper measurement. For in vivo bioluminescence detection, micewere anesthetized using 2% isofluorane/air mixture and injectedintraperitoneally with a single dose (150 mg/kg) D-luciferin. Photoncounts/sec were acquired before and 6 hrs post-TRAIL treatment (FIG.11A) using IVIS imaging system (Caliper Life Sciences). Fold induction(FIG. 11B) was calculated by normalizing post treatment values topre-treatment values per mouse.

The data demonstrates that the mutant thermostable biosensors of thepresent invention are extremely sensitive as 100 fold bioluminescenceactivation upon TRAIL treatment was seen in the mouse xenograft model.D54-MG reporter xenografted nude mice were treated with 8 mg/kg ofTRAIL. Photon counts/sec were acquired pre- and post-treatment (FIG.11). The fold induction was calculated by normalizing post treatmentvalues to pre treatment values per mouse (FIG. 11B). FIG. 11C shows atable depicting the average photon counts/sec at baseline and 6 hrs posttreatment in addition to fold changes achieved with different biosensorversions.

Example VIII Imaging of Cell Death in Breast Bone Metastasis

To demonstrate the use of the thermostable caspase-3 biosensors todetect cell death in animals, 100,000 MDA-MB231/1833 cells (“1833”;breast cancer cell line) stably expressing TL-CP233-Caspase-3 biosensor(derived as described in Example VI for glioma cells) were implantedinto the tibia of nude mice. Tumor growth was followed by MRI and TRAILtreatment was initiated when the tumor reached 5-15 mm³.

For in vivo bioluminescence detection, mice were anesthetized using 2%isofluorane/air mixture and injected intraperitoneally with a singledose (150 mg/kg) D-luciferin. Photon counts/sec were acquired before and6 hrs post-TRAIL treatment or as indicated in FIG. 12A-D using IVISimaging system (Caliper Life Sciences). Fold induction (FIG. 11B) wascalculated by normalizing post treatment values to pre-treatment valuesper mouse.

In FIG. 12A, intratibial implanted MDA-MB231/1833 cells stablyexpressing TL-CP233-Caspase-3 were treated with TRAIL (200 ng/mL) andimaged every hour for 10 consecutive min. Fold induction was calculatedby normalizing data to pre-treatment value. In FIG. 12B, Z factors werecalculated as described in Zhang et al (Biomol Screen. 4:67-73. 1999)for every time point, and an average Z factor of 0.82 sufficed assaysuitability for high-throughput screening. In FIG. 12C, representativeimages taken at the indicated time points of intratibial implantedTL-CP233-Caspase-3 stably expressing MDA-MB231/1833 cells with thephotons/sec. In FIG. 12D, fold induction of xenografted animals testedtreated with TRAIL. This data highlights the usefulness of thethermostable biosensor for imaging cell death dynamically and over timein mouse models.

Example IX Utility of the Thermostable Caspase Biosensor inHigh-Throughput Screening

To demonstrate the utility of the thermostable biosensors forhigh-throughput screening (HTS), the MDA-MB231/1833 (“1833”) cellsstably expressing TL-CP233-Caspase-3 from Example VIII were used toscreen compounds in the NIH Clinical Collection Biofocus and TimTecKinase Inhibitor libraries.

TL-CP233-Caspase-3 MDA-MB231/1833 cells were seeded at 10,000 cells/wellin a 96-well plate. Forty-eight hrs post-seeding, the media was changedto CO₂ Independent Media containing 1% GloSensor cAMP Reagent (Promega;Cat. No. E1290) and incubated for 0-23 hrs with compound at a finalconcentration of 10 μM. A total of 483 compounds in the NIH ClinicalCollection and 80 kinase inhibitors from the TimTec collection weretested. The addition of media and compound library was performed using aTitertek Multidrop Microplate Dispensor (ThermoFisher Scientific).Relative luminescence was calculated by normalizing values of compoundtreated wells to untreated wells. (FIGS. 13A and 13C). FIG. 13A showsthe relative luminescence upon compound treatment (max) from compoundsin the NIH Clinical Collection Biofocus Library. FIG. 13C shows therelative luminescence upon compound treatment (max) from compounds inthe TimTec Kinase Inhibitor Library. Maximum values reaching above 4were considered significant. Heat maps were generated usingbioinformatics toolbox of Matlab Software and show correlation ofbiosensor activation over time. (FIGS. 13B and 13D). The Z-factor wascalculated as previously described in Example VIII.

Due to the ability of repeated imaging of the thermostable biosensor,dynamics of apoptosis in response to various drugs could be imaged. Thisallowed for the identification of interesting death inducing compoundsin the otherwise chemoresistant 1833 breast cancer cell line.

Example X Purification of MMP-2 Sensor

The matrix metalloproteinases (MMP) are a homologous group of zincenzymes that participate in the breakdown of the major proteincomponents of the extracellular matrix. Five major MMP have beenidentified in humans and implicated in connective tissue turnover anddestruction. These include the fibroblast-type and neutrophil-typeinterstitial collagenases that hydrolyze the type I, II, and IIIcollagens that make up the majority of the matrix. Fibroblastcollagenase also hydrolyzes native type VII and X collagens. The MMP aresometimes referred to by a numerical code in which the fibroblast-typeand neutrophil-type collagenases are designated MMP-1 and MMP-8,respectively. A 72-kDa gelatinase (MMP-2) is produced by proliferatingfibroblasts and tumor cells, while a distinct 92-kDa gelatinase (MMP-9)is produced by neutrophils, macrophages, and certain transformed cells.

The MMP-2 sensor (SEQ ID NOs:69 and 70) used herein contains the 1A5variant backbone and the human MMP-2 recognition site, PLGMWSR (SEQ IDNO:75). In addition, the MMP-2 sensor contains two purification tags: aGST tag on the N-terminus of the sensor that is separated from thesensor region by a TEV protease site (for removal of the GST tag fromthe purified MMP-2 sensor) and a 5×HQ (HQHQHQHQHQ; SEQ ID NO:79) tag onthe C-terminus of the sensor.

Purification of the MMP-2 sensor was performed as follows using His andGST purification.

1. 2-5 mL cultures of E. coli KRX cells (Promega) containing the MMP-2sensor were grown in LB/ampicillin with shaking at 37° C.

2. Each culture was diluted 1:100 in 1 L LB with 0.05% rhamnose and0.05% glucose.

3. Incubated at 25° C. for 18-20 hrs.

4. Cells were harvested by centrifugation at 5000 g for 5 min (split 1 Linto 2-500 mL aliquots), cell paste weight was determined, and placed at−20° C. overnight.

5. One of the 2 cell pastes was resuspended with 30 mL lysis buffer (8.5mL/g cell paste; 50 mM NaH₂PO₄, 300 mM NaCl, 10 mM Imidazole and pH to8.0 with NaOH). 1 mg/mL lysozyme was added, and the resuspension wasincubated on ice, with inverting occasionally, for 30 min.

6. The lysis solution was sonicated at power 6.0 for 2 min (5 sec on, 5sec off). 100 μl of the sample was saved as “total” sample.

7. The lysis solution was spun at 16,000 g for 20 min. 100 μl of thesample was saved as “soluble” sample.

8. 1 mL 50% Ni-NTA resin (Qiagen; pre-washed in lysis buffer) per 6 mLlysate (5 mLs total) was added and mixed at 4° C. for 1 hr.

9. Sample was spun at 700 rpm on a tabletop centrifuge for 2 min. 100 μlof the sample was saved as “flowthrough” sample with the supernatantdiscarded.

10. The resin was washed in 40 mL lysis buffer, mixed at 4° C. for 5min, spun at 700 rpm on a tabletop centrifuge for 2 min, and thesupernatant was discarded.

11. The resin was then washed in 40 mL wash buffer (lysis buffer with 20mM Imidazole), spun at 700 rpm on a tabletop centrifuge for 2 min, andthe supernatant was discarded.

12. 10 mL of wash buffer was added and mixed, and the supernatant wasadded to an empty column.

13. The column was washed with 50 mL wash buffer and 100 μL resin wasremoved and saved.

14. The sensor was eluted from the column with 10 mL elution buffer(lysis buffer with 250 mM Imidazole) with 0.5 mL fractions collected anddirectly assayed using the Bradford Assay.

15. 100 μL of the resin was removed and saved, the elution fractionswere combined, and the combined fraction was diluted to 10 mL in lysisbuffer.

16. The combined fraction was dialyzed (1 hr with 1 L twice) in GSTbinding/wash buffer (1×PBS).

17. The dialyzed protein was added to 5 mL glutathione-sepharose resinslurry (GE Cat #17-0756-01) prewashed in GST binding/wash buffer) andwas incubated for 1 hr at 4° C.

18. The resin mixture was spun at 700 rpm in a tabletop centrifuge for 2min, 100 μL was saved as “flowthrough”, and the supernatant wasdiscarded.

19. The resin was added to an empty column, washed with 50 mL GSTbinding/wash buffer, and 100 μL was removed.

20. The protein was eluted with elution buffer (1×PBS buffer with 10 mMreduced Glutathione). 0.5 mL fractions were collected and directlyassayed using the Bradford assay. 100 μL of resin was removed and saved.

21. The saved fractions (“total”, “soluble”, “flowthrough”(his), Hisresin before elution, His resin after elution, after dialysis sample,flowthrough (GST), GST resin before elution, GST resin after elution andGST fractions) were analyzed on an SDS-PAGE gel (FIG. 14).

22. The GST fractions were combined and dialyzed in storage buffer (50mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT, 50% glycerol).

To demonstrate that the purified MMP-2 sensor can detect MMP-2, purifiedMMP-2 sensor (1.3 mg/mL) was diluted in buffer (50 mM Tris-HCl pH 7.5,150 mM NaCl, 10 mM CaCl₂, and 0.05% Brij35) as described in FIG. 15.Activated MMP-2 (10 ng/μL; Anaspec) was added to the diluted MMP-2sensor as described in FIG. 15. Total volume of the final reactionmixture was 50 μL. The mixture was incubated at 37° C. for 1 hr.Bright-Glo Assay Reagent (Promega) was prepared according to themanufacturer's instructions, and 50 μL added to the mixture.Luminescence was read immediately on a GloMax MultiPlus luminometer.

FIG. 15 presents the fold increase over control (background from MMP-2sensor). The results demonstrate that a purified MMP-2 sensor accordingto the present invention can be used to detect as little as 0.75 ngprotein.

To further demonstrate the sensitivity of the purified MMP-2 sensor, thefluorogenic SensoLyte 520 MMP-2 Assay (Anaspec) was also used to detectMMP-2 protein. The assay was performed according to the manufacturer'sinstructions. Fluorescence was detected on a Tecan fluorometer at Ex490nm/Em520 nm. FIGS. 16A-B report detection of 1.5 ng MMP-2 protein usingthe SensoLyte assay.

Example XI Cell-Free Expression of CBS

To demonstrate that the protease biosensor of the present invention caneffectively and efficiently be cleaved by exogenous protease in acell-free environment, the CBS variant 1A5 was expressed in wheat germextract and used to detect Caspase-3.

The CBS variant 1A5 was cloned into the vector pFN19K HaloTag® (PromegaCat. No. G1841) to generate a CBS-HaloTag® (CBS-HT) fusion protein (SEQID NOs:71 and 72). 20 μl (8 μg) of the CBS-HT vector was added to 30 μlTnT® SP6 High-Yield Wheat Germ Expression System (Promega Cat. No.L3261) and incubated at 25° C. for 90 min.

For the CBS-HT caspase-3 cleavage reaction, one volume of the expressionreaction was incubated with an equal volume of either an E. coli lysatecontaining caspase-3 or C(3) Lysis Buffer (0.8× FastBreak (Promega Cat.No. V857A), 10 mM DTT, 0.1% CHAPS, 0.8 mg/mL Lysozyme, 3 U/μL RQ1 DNase(Promega Cat. No. M610A)) and incubated for 60 min at room temperature.The E. coli lysate containing caspase-3 was prepared from KRX cellsoverexpressing recombinant caspase-3. Briefly, KRX cells weretransformed with pTS1k:caspase-3(T/S). A starter culture (50 mL, LBBroth) was inoculated from a single colony and grown for 17-22 hrs at37° C. with shaking (275 rpm). The starter culture was diluted (1:50)into fresh media and growth was continued for an additional 3 hrs. Theincubation temperature was then lowered to 25° C. and, after 15 min,expression of caspase-3 was initiated by addition of rhamnose (0.2%final concentration). After 2 hr, cells were collected bycentrifugation, re-suspended in 50 mL C(3) Lysis Buffer and incubated atambient temperature (i.e. 22-24° C.) for 10 min. The lysate wasclarified by centrifugation (20,000×g for 20 min at 4° C.) and used asthe Caspase-3 source.

Cleavage of CBS-HT by Caspase-3 was detected in two different ways: bySDS-PAGE analysis and luminescence detection. For SDS-PAGE analysis,cleavage reaction samples were first labeled with the fluorescent markerCA-TAM (chloro alkane-TAMRA ligand (Promega Cat. No. G825A). 20 μl ofthe sample was added to 20 μL CA-TAM (diluted 1:100 in buffer (1×PBS,0.05% IGEPAL)) and incubated for 30 min at room temperature. To thissample, 40 μL SDS-PAGE Loading Buffer (120 mM Tris Buffer (pH7.4), 1%SDS, 25.2% Glycerol, 1.5 mM Bromophenol Blue, 100 mM DTT) was added. Theresulting solution was incubated at 65° C. for 30 min. 10 μL was loadedonto an SDS-PAGE gel. As a control, 0.60, 0.15 and 0.03 mg/mL HT:GST(HaloTag®-GST (Promega Cat. No. G449A) fusion was also loaded onto thegel (FIG. 17). After electrophoresis, CA-TAM labeled species weredetected by fluorescent imaging (ex:532, Em:580; FIG. 17). For detectionvia luminescence, 40 μL of the cleavage reaction samples were added to60 μL buffer (50 mM HEPES (pH 7.5) and 100 μL Bright-Glo assay reagent.Luminescence was detected as previously described (Table 7).

TABLE 7 WG(HY) MIN BASAL INDUCED RESPONSE 5 10,494 4,626,173 441

Example XII Immobilization of CBS

To demonstrate that the protease biosensor of the present invention whenexpressed in a cell-free environment maintains activity when immobilizedon a solid support, the CBS-HT fusion expressed in wheat germ extract(Example XI) was immobilized to a solid support (resin and plate) andused to detect Caspase-3.

For immobilization to a resin (FIG. 18A), HaloLink resin (25% slurry,Promega Cat. No. G1912) was first equilibrated with HTPB Buffer (50 mMHEPES (pH 7.5), 150 mM NaCl, 1 mM DTT and 0.5 mM EDTA). Resin from onevolume of slurry was collected by centrifugation (5 min at 1000×g), andthe storage buffer was removed. The resin pellet was re-suspended in 2volumes of HTPB and mixed. This process was repeated for a total ofthree HTPB washes. 100 μL of the CBS-HT fusion from Example XI (cellfree expression reaction) was mixed with 25 μL washed resin (50% slurryin HTPB) and incubated overnight with mixing at 4° C. Incubation wascontinued at ambient temperature for 2 hrs. After incubation, the resinwas washed to remove un-bound CBS. The resin was split into two 50 μlaliquots, and each aliquot was washed 3 times with HTPB. The final resinpellets were re-suspended in 50 μL HTPB.

For the CBS caspase-3 cleavage reaction, 20 μl of the washed resin wasmixed with 20 μL of either E. coli lysate containing caspase-3 or C(3)Lysis Buffer (as in Example XI) and incubated for 30 min at ambienttemperature. 60 μl HEPES pH 7.5 was added to the samples, followed bythe addition of 100 μl Bright-Glo Assay Reagent. Luminescence wasdetected as previously described (Table 8 and FIG. 19).

TABLE 8 BASAL INDUCED RESPONSE BLANK 10 70 7 AS 29,553 5,162,108 175 FT3,170 232,117 73 RESIN 11,219 1,635,259 146

For immobilization to a plate (FIG. 18B), a microtiter plate wasprepared containing HaloTag® ligand for immobilizing the CBS-HT fusionprotein. Briefly, bicarbonate buffer pH 8.5 (100 μL), containingamine-PEG 2000-Cl alkane HaloTag® ligand (0.25 mM (final concentration;PBI 3961 and methoxy-PEG-NH₂ (0.75 mM final concentration) were added towells of a NHS microtiter plate and incubated for 1 hr at roomtemperature. The wells were then washed 3 times with PBS containing0.05% Tween-20. After washing, 50 mM ethanolamine was added, and theplates were incubated for 30 min at room temperature and washed again 3times with PBS containing 0.05% Tween-20. The plate was then stored at4° C. with PBS containing 0.05% Tween-20 in each well. For the assay,the wells were washed 3 times with 200 μl HPTB. 50 μl CBS-HT cell-freeexpression reaction (Example XI) was added to the wells, and incubatedovernight at 4° C. Following incubation, the plate was washed 3 timeswith 200 μl PBSI (1×PBS with 0.05% IGEPAL). 100 μL E. coli lysatecontaining caspase-3 or C(3) lysis buffer (described above) were addedand the plate was incubated for 60 min with mixing at room temperature.The wells were then washed with 100 μl PBSI. 100 μL HEPES pH 7.5 and 100μl Bright-Glo Assay Reagent were added and luminescence was detected aspreviously described (Table 9).

TABLE 9 MIN BASAL INDUCED RESPONSE 0 66 1,226 19 1 16 1,636 102 2 1062,267 21 3 66 2,147 33 4 126 2,297 18 5 126 2,857 23 6 76 3,107 41 7 563,377 60 8 136 3,037 22 9 116 3,667 32

Example XIII CBS Expression in E. coli

To demonstrate that the protease biosensor of the present invention canbe expressed and function in E. coli, the CBS variant 1A5 was expressedin E. coli and used to detect Caspase-3.

The CBS variant 1A5 was cloned into a bacterial expression vector(pFNA:HQ(5×):CBS:HT(7); SEQ ID NOs:73 and 74) containing HaloTag®(C-terminal to the CBS) and a 5×HQ tag (N-terminal to CBS). The fusionprotein was expressed in E. coli as follows: E. coli (KRX) wastransformed with the vector. A starter culture (50 mL, LB Broth) wasinoculated from a single colony and grown for 17-22 hrs at 37° C. withshaking (275 rpm). The starter culture was diluted (1:50) into inductionmedia (500 mL, LB Broth with 0.05% glucose and 0.02% rhamnose) andgrowth was continued for another 17-22 hrs at 25° C. with shaking (275rpm). The culture was divided into two 250 mL aliquots, and cells werecollected by centrifugation (5,000×g for 20 min at 4° C.). One cellpellet was re-suspended in Lysis Buffer (25 mL, 50 mM HEPES (pH 7.5),0.2× FastBreak, 2 mM DTT, 0.05% CHAPS, 50 mM Arginine, 50 mM Glutamicacid, 0.2 mg/mL Lysozyme, 10 U/mL RQ1 DNase, and Protease Inhibitors(Beckton/Dickenson Cat. No. 544779)) and incubated on ice for 30 min.After incubation, the sample was sonicated (Misonix Sonicator-3000, 4min total, 5 sec on, 5 sec rest, Power Setting 5). The crude lysate wasclarified by centrifugation (20,000×g for 20 min at 4° C.), and thesupernatant (cleared lysate) was used as the CBS source. For thecaspase-3 cleavage reaction, 20 μl of the cleared lysate was mixed with20 μL E. coli lysate expressing caspase-3 (Example XII) and incubated atroom temperature for 30 min. 60 μL HEPES pH 7.5 was added, followed bythe addition of 100 μL Bright-Glo Assay Reagent. Luminescence wasdetected as previously described above (FIG. 20).

Example XIV Purification of CBS from E. coli

To demonstrate the ability to purify a functional protease biosensor ofthe present invention from E. coli, the CBS expressed in Example XIIIwas purified using HisLink (Promega Cat. No.V8821)) columnchromatography according to the manufacturer's instructions. Briefly, 25mL of cleared lysate was made 0.5 M in NaCl (final concentration) andapplied to 2 mL of settled HisLink resin that had been equilibrated withBinding Buffer (100 mM HEPES (pH7.5), 10 mM Imidazole, 500 mM NaCl). Theresin was washed with 12 mL of Binding Buffer followed by washing withElution Buffer (100 mM HEPES (pH 7.5), 1000 mM Imidazole). 1.75 mLfractions were collected.

For SDS-PAGE gel analysis, samples were labeled with CA-TAM and analyzedas described previously (FIG. 21A). For caspase-3 cleavage reaction, 20μl of each sample was mixed with 20 μL E. coli lysate expressingCaspase-3 (Example XII) and incubated at room temperature for 30 min. 60μL HEPES pH 7.5 was added, followed by the addition of 100 μL Bright-GloAssay Reagent. Luminescence was detected as previously described above(Table 10 and FIG. 21B).

TABLE 10 HSS N5M WASH E1 E2 E3 E4 E5 BASAL 301,304 176,259 7,734 4,221193,268 18,454 1,130 270 INDUCED 12,875,742 5,822,003 3,706,5412,951,899 40,913,496 3,287,059 215,580 48,068 RESPONSE 43 33 479 699 212178 191 178

Example XV Immobilization of E. coli Expressed CBS

To demonstrate that the protease biosensor expressed in E. coli can beimmobilized to a solid support while maintaining the ability to detectprotease, purified HQ:CBS:HT (5×HQ tag:CBS: HaloTag) was immobilized onHaloLink resin and HaloLink plates and assayed for activation byCaspase-3. For immobilization on HaloLink resin, 100 μL of purifiedHQ:CBS:HT was added to 30 μL of settled HaloLink resin (pre-equilibratedwith HTPB as described previously) and incubated for 2 hrs at ambienttemperature. The resin was washed 3 times with 300 μL HTPB, and thefinal resin pellet was re-suspended in 300 μL HTPB. 50 μL washed resinwas added to 50 μL E. coli Lysate containing Caspase-3 or 50 μL C(3)Lysis Buffer and incubated for 30 min at ambient temperature. 100 μL ofBright-Glo Assay Reagent was added and luminescence was detected aspreviously described (Table 11 and FIG. 22).

For immobilization on HaloLink plates, 100 μL of purified HQ:CBS:HT wasadded to a microtiter plate containing immobilized HaloTag® ligand(Example XII). The plate was incubated at room temperature with mixingfor 2 hrs. The plate was then washed 3 times in 1×PBSI (1×PBS with 0.05%IGEPAL) and incubated with 100 μL of E. coli Lysate containing Caspase-3(prepared as described previously) for 30 min at room temperature. 100μL Bright-Glo Assay Reagent was added, and luminescence detected aspreviously described (Table 11 and FIG. 22).

TABLE 11 HisLink HaloLink-Plate BASAL 193,268 6,880 INDUCED 40,913,496661,597 RESPONSE 212 96

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification, thisinvention has been described in relation to certain embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsherein may be varied considerably without departing from the basicprinciples of the invention.

Thus, the invention provides, among other things, a modifiedcircularly-permuted thermostable luciferase biosensor with enhancedresponse to a target molecule. Various features and advantages of theinvention are set forth in the following claims.

1. A polynucleotide encoding a biosensor polypeptide comprising amodified circularly-permuted thermostable luciferase and a linkerlinking the C-terminal portion of the thermostable luciferase to theN-terminal portion of the thermostable luciferase, the modifiedcircularly-permuted thermostable luciferase is modified relative to aparental circularly-permuted thermostable luciferase, the linkercomprising a sensor region capable of interacting with a target moleculein a cell wherein the modified circularly-permuted thermostableluciferase has an enhanced response after interaction of the biosensorwith the target molecule relative to at least one of: i) the parentalcircularly-permuted thermostable luciferase in the presence of thetarget molecule, or ii) the modified circularly-permuted thermostableluciferase in the absence of the target molecule.
 2. The polynucleotideof claim 1, wherein the modified circularly-permuted thermostableluciferase comprises a substitution at an amino acid corresponding toposition 507 of SEQ ID NO:
 2. 3. (canceled)
 4. The polynucleotide ofclaim 1, wherein the modified circularly-permuted thermostableluciferase comprises a substitution at an amino acid corresponding toposition 503 of SEQ ID NO:
 2. 5. (canceled)
 6. The polynucleotide ofclaim 1, wherein the modified circularly-permuted thermostableluciferase comprises a substitution at an amino acid corresponding toposition 471 of SEQ ID NO:
 2. 7. (canceled)
 8. The polynucleotide ofclaim 1, wherein the modified circularly-permuted thermostableluciferase comprises a substitution at an amino acid corresponding toposition 193 of SEQ ID NO:
 2. 9. (canceled)
 10. The polynucleotide ofclaim 1, wherein the modified circularly-permuted thermostableluciferase comprises amino acid substitutions corresponding to 1471T,S503G, T507I, and S193P of SEQ ID NO:2.
 11. The polynucleotide of claim1, wherein the modified circularly-permuted thermostable luciferasecomprises amino acid substitutions corresponding to I471T, S503G, andT507I of SEQ ID NO:2.
 12. The polynucleotide of claim 1, wherein themodified circularly-permuted thermostable luciferase comprises aminoacid substitutions corresponding to S503G, T507I, and S193P of SEQ IDNO:2.
 13. The polynucleotide of claim 1, wherein the modifiedcircularly-permuted thermostable luciferase comprises an amino acidsubstitution corresponding to T507I of SEQ ID NO:2.
 14. Thepolynucleotide of claim 1, wherein the modified circularly-permutedthermostable luciferase comprises a substitution of at least one aminoacid corresponding to positions 5, 17, 21, 23, 26, 39, 44, 51, 81, 101,103, 110, 114, 115, 119, 123, 126, 128, 133, 137, 186, 191, 192, 193,196, 208, 211, 214, 226, 228, 230, 233, 264, 273, 275, 286, 287, 294,295, 297, 302, 303, 304, 306, 308, 309, 313, 324, 329, 331, 343, 348,353, 364, 374, 385, 389, 409, 420, 426, 427, 428, 431, 449, 456, 460,461, 465, 466, 468, 471, 473, 482, 484, 485, 489, 493, 494, 497, 503,507, 509, 510, 513, 516, 517, 521, 522, 523, 526, 530, 533, 536, 537,542, or 543 of SEQ ID NO: 2, or combination thereof.
 15. Thepolynucleotide of claim 1, wherein the thermostable luciferase iscircularly-permuted in a region corresponding to residues 2 to 12,residues 32 to 53, residues 70 to 88, residues 102 to 126, residues 139to 165, residues 183 to 203, residues 220 to 247, residues 262 to 273,residues 303 to 313, residues 353 to 408, residues 485 to 495, orresidues 535 to 546 of a firefly luciferase.
 16. The polynucleotide ofclaim 1, wherein the biosensor comprises a protease recognition site, akinase recognition site, an antibody binding site, a metal binding site,an ion binding site, a cyclic nucleotide binding site or a nucleotidebinding site.
 17. The polynucleotide of claim 1, wherein the biosensorcomprises a protease recognition site.
 18. The polynucleotide of claim17, wherein the protease recognition site is selected from the groupconsisting of a caspase-3 recognition site, a caspase-8 recognitionsite, an enterokinase recognition site, a prostate serum antigenrecognition site, a SARS viral protease recognition site, a TEV proteaserecognition site, a Granzyme B recognition site, a MMP recognition site,and a rhinovirus protease recognition site.
 19. The polynucleotide ofclaim 1, wherein the thermostable luciferase is modified from awild-type luciferase from a species selected from the group consistingof Luciola cruciata, Luciola lateralis, Pyrocoelia miyako, Lampyrisnoctiluca, Photuris pennsylvanica, Phengodes sp., Luciola mingrelica,and Photinus pyralis.
 20. The polynucleotide of claim 1, wherein thethermostable luciferase is a firefly luciferase.
 21. The polynucleotideof claim 20, wherein the thermostable luciferase has at least 95% aminoacid identity to SEQ ID NO:2.
 22. The polynucleotide of claim 20,wherein the thermostable luciferase has at least 90% amino acid identityto SEQ ID NO:4.
 23. The polynucleotide of claim 20, wherein thethermostable luciferase has at least 95% amino acid identity to SEQ IDNO:4.
 24. The polynucleotide of claim 1, wherein the linker comprisesthe sensor region DEVD.
 25. The polynucleotide of claim 1, wherein thelinker is SSDEVDGSSG (SEQ ID NO:52), SSGSDEVDGSLSSG (SEQ ID NO:53),SDEVDGSL (SEQ ID NO:54), or DEVDG (SEQ ID NO: 55).
 26. A polynucleotideencoding a polypeptide according to SEQ ID NO:6.
 27. A vector comprisingthe polynucleotide of claim
 1. 28. (canceled)
 29. A cell comprising thepolynucleotide of claim
 1. 30. A non-human transgenic animal comprisingthe cell of claim
 29. 31.-33. (canceled)
 34. A kit comprising thepolynucleotide of claim
 1. 35.-70. (canceled)
 71. A cell comprising thevector of claim
 27. 72. A kit comprising the vector of claim 27.